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Sample records for inhibits phosphatidylinositol turnover

  1. Phosphatidylinositol turnover in mitogen-activated lymphocytes. Suppression by low-density lipoproteins

    PubMed Central

    Hui, David Y.; Harmony, Judith A. K.

    1980-01-01

    Low-density (LD) lipoproteins inhibit phytohaemagglutinin-enhanced turnover of phosphatidylinositol in human peripheral lymphocytes. Turnover was assessed by 32P incorporation into phospholipids and by loss of 32P from [32P]phosphatidylinositol. Inhibition of lipid turnover by LD lipoproteins is not the result of a change in the amount of phytohaemagglutinin required for maximum cellular response. Neither phytohaemagglutinin nor LD lipoproteins influence 32P incorporation into phosphatidylethanolamine and phosphatidylcholine during the first 60min after mitogenic challenge. The extent of inhibition of phosphatidylinositol turnover by LD lipoproteins depends on the concentration of LD lipoproteins present in the incubation medium: 50% of maximum inhibition occurs at a low-density-lipoprotein protein concentration of 33μg/ml and maximum inhibition occurs at low-density-lipoprotein protein concentrations above 100μg/ml. Phytohaemagglutinin stimulates 32P incorporation into phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. However, LD lipoproteins abolish 32P incorporation into phosphatidylinositol without affecting incorporation into phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The ability of LD lipoproteins to inhibit phytohaemagglutinin-induced phosphatidylinositol turnover is mimicked by EGTA. Furthermore, inhibition of LD lipoproteins by phytohaemagglutinin-induced 32P incorporation into phosphatidylinositol correlates directly with inhibition by LD lipoproteins of Ca2+ accumulation. These results suggest that Ca2+ accumulation and turnover of phosphatidylinositol are coupled responses in lymphocytes challenged by mitogens. The step in phosphatidylinositol metabolism that is sensitive to LD lipoproteins and, by inference, that is coupled to Ca2+ accumulation is release of [32P]phosphoinositol from phosphatidylinositol. PMID:6796039

  2. Adenosine (ADO) released during orthodromic stimulation of the frog sympathetic ganglion inhibits phosphatidylinositol turnover (PI) associated with synaptic transmission

    SciTech Connect

    Curnish, R.; Bencherif, M.; Rubio, R.; Berne, R.M.

    1986-03-05

    The authors have previously demonstrated that /sup 3/H-purine release was enhanced during synaptic activation of the prelabelled frog sympathetic ganglion. In addition, during orthodromic stimulation, there is an increased /sup 3/H-inositol release (an index of PI) that occurs during the poststimulation period and not during the period of stimulation. They hypothesized that endogenous ADO inhibits PI turnover during orthodromic stimulation. To test this hypothesis (1) they performed experiments to directly measure ADO release in the extracellular fluid by placing the ganglion in a 5 ..mu..l drop of Ringer's and let it come to equilibrium with the interstitial fluid, (2) they destroyed endogenous ADO by suffusing adenosine deaminase (ADA) during the stimulation period. Their results show (1) orthodromic stimulation increases release of ADO into the bathing medium, (2) ADA induced an increase of PI during the stimulation period in contrast to an increase seen only during the poststimulation period when ADA was omitted. They conclude that there is dual control of PI during synaptic activity, a stimulatory effect (cause unknown) and a short lived inhibitory effect that is probably caused by adenosine.

  3. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    SciTech Connect

    Boura, Evzen Nencka, Radim

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  4. Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: stimulatory effects of agents that enhance influx of sodium ions.

    PubMed Central

    Gusovsky, F; Hollingsworth, E B; Daly, J W

    1986-01-01

    Norepinephrine and carbamoylcholine stimulate accumulation of [3H]inositol phosphates from [3H]inositol-labeled guinea pig cerebral cortical synaptoneurosomes through interaction with alpha 1-adrenergic and muscarinic receptors, respectively. In addition to such agonist, a variety of natural products that affect voltage-dependent sodium channels can markedly stimulate accumulation of [3H]inositol phosphates. These include alkaloids that activate sodium channels, such as batrachotoxin, veratridine, and aconitine; peptide toxins that alter activation or slow inactivation of sodium channels, such as various scorpion toxins from Leiurus, Centruroides, and Tityus species; and agents that cause repetitive firing of sodium channel-dependent action potentials, such as pyrethroids and pumiliotoxin B. Ouabain, and agent that will increase accumulation of internal sodium by inhibition of Na+, K+-ATPase, also stimulates formation of [3H]inositol phosphates, as does monensin, a sodium ionophore. Tetrodotoxin and saxitoxin, specific blockers of voltage-dependent sodium channels, prevent or reduce the stimulatory effects of sodium channel agents and ouabain on phosphatidylinositol turnover, while having lesser or no effect, respectively, on receptor-mediated or monensin-mediated stimulation. Removal of extracellular sodium ions markedly reduces stimulatory effects of sodium channel agents, while removal of extracellular calcium ions with EGTA blocks both receptor-mediated and sodium channel agent-mediated phosphatidylinositol turnover. The results provide evidence for a hitherto unsuspected messenger role for sodium ions in excitable tissue, whereby neuronal activity and the resultant influx of sodium will cause activation of phospholipase systems involved in hydrolysis of phosphatidylinositols, thereby generating two second messengers, the inositol phosphates, which mobilize calcium from internal stores, and the diacylglycerols, which activate protein kinase C. PMID:2422664

  5. Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity

    SciTech Connect

    Chen, Qiuyun; Boss, W.F. )

    1991-05-01

    The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP{sub 2}), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes. In this paper, the authors show the effect of various concentrations of phosphatidyinositol, PIP, and PIP{sub 2} on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP{sub 2} at concentrations at 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.

  6. Phosphatidylinositol inhibits respiratory syncytial virus infection

    PubMed Central

    Numata, Mari; Kandasamy, Pitchaimani; Nagashima, Yoji; Fickes, Rachel; Murphy, Robert C.; Voelker, Dennis R.

    2015-01-01

    Respiratory syncytial virus (RSV) infects nearly all children under age 2, and reinfection occurs throughout life, seriously impacting adults with chronic pulmonary diseases. Recent data demonstrate that the anionic pulmonary surfactant lipid phosphatidylglycerol (PG) exerts a potent antiviral effect against RSV in vitro and in vivo. Phosphatidylinositol (PI) is also an anionic pulmonary surfactant phospholipid, and we tested its antiviral activity. PI liposomes completely suppress interleukin-8 production from BEAS2B epithelial cells challenged with RSV. The presence of PI during viral challenge in vitro reduces infection by a factor of >103. PI binds RSV with high affinity, preventing virus attachment to epithelial cells. Intranasal inoculation with PI along with RSV in mice reduces the viral burden 30-fold, eliminates the influx of inflammatory cells, and reduces tissue histopathology. Pharmacological doses of PI persist for >6 h in mouse lung. Pretreatment of mice with PI at 2 h prior to viral infection effectively suppresses inflammation and reduces the viral burden by 85%. These data demonstrate that PI has potent antiviral properties, a long residence time in the extracellular bronchoalveolar compartment, and a significant prophylaxis window. The findings demonstrate PG and PI have complementary roles as intrinsic, innate immune antiviral mediators in the lung. PMID:25561461

  7. Phosphatidylinositol turnover is associated with human natural killer cell activation by tumor target cells

    SciTech Connect

    Steele, T.A.; Brahmi, Z.

    1986-03-01

    Natural Killer (NK) cell activity has been shown to be a binding-dependent event leading to the destruction of various targets. This suggests a possible role for plasma membrane phospholipid turnover in coupling a receptor-mediated binding event with transduction of a intracellular signal to result in the activation of the effector cell. Currently, phosphatidylinositol (PI) turnover is implicated in several immune cell systems. Therefore, in this study, the authors examined phospholipid turnover in human NK cells upon exposure to a sensitive (K562) and a resistant (YAC-1) target cell (TC). NK cell membrane phospholipids were labelled with Phosphorus-32 (/sup 32/P) and, following stimulation, were extracted and run on silica gel thin-layer chromatography. Labelled phospholipids were visualized by autoradiography then scraped and counted in a liquid scintillation counter. A 2.5 fold increase in label incorporation into PI relative to controls was shown to occur when NK cells were stimulated by K562 for 2 hours. In contrast, no increased labelling of PI relative to controls was noted when NK cells were stimulated by YAC-1 for the same period of time. No change in incorporation of /sup 32/P into phosphatidylcholine or phosphatidylethanolamine occurred in either set of conditions. These results suggest that PI turnover may be an early activation event in NK cells following binding of K562.

  8. Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes.

    PubMed Central

    Hegewald, H; Müller, E; Klinger, R; Wetzker, R; Frunder, H

    1987-01-01

    In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+. PMID:2821996

  9. Characteristics of the norepinephrine-stimulated phosphatidylinositol turnover in rat pineal cell dispersions

    SciTech Connect

    Hauser, G.; Smith, T.L.

    1981-10-01

    Dispersed rat pineal cells can be used for the study of the phosphatidylinositol effect. The response to ( - )-norepinephrine of the incorporation of 32Pi into phospholipids is linear with time and cell concentration, stereospecific, and mediated through alpha-1-adrenergic receptors. Na+ in the incubation medium is obligatory for labeling of phosphatidylinositol and phosphatidylcholine by 32P. In the absence of K+, incorporation of 32P is drastically lowered and no stimulation by norepinephrine occurs. Rb+ can replace K+. Omission of Ca2+ or substitution with Sr2+ preferentially lowers incorporation of radioactivity into phosphatidylcholine. Mg2+ is not required for basal or stimulated labeling.

  10. Mitogenic stimuli and phosphatidylinositol (PI) turnover in cultured 3T3 fibroblasts

    SciTech Connect

    Kohler, C.; Petersen, R.

    1986-03-01

    The hydrolysis of PI and polyphosphoinositides by phopholipase C is an early and rapid response to cell activation by a variety of neurotransmitters, hormones, growth factors and pharmacological agonists. The authors have examined the role of PI turnover and the generation of second messengers (diacylglycerol and inositol trisphosphate) in the mitogenic response of cultured Balb/c and Swiss 3T3 cells to polypeptide growth factors. Cells were prelabelled with /sup 3/H inositol for 18-20 hours, washed and suspended in Herpes + Li/sup +/ buffer, and stimulated with platelet-derived growth factor (PDGF), vasopressin, insulin, and other growth factors. PI turnover was measured as the increase in total inositol phosphate (IP) production. IP1, IP2, and IP3 were characterized by sequential elution from a Dowex column. Partially purified PDGF produced a 2-4 fold stimulation of total IP production. This was seen as early as 30 seconds after stimulation and increased for up to 1-2 hours. Balb/c cells were more sensitive than Swiss cells to the mitogenic and PI effects of PDGF. Other mitogenic stimuli had differential effects on PI turnover. Vasopressin (4-400 ng/ml) markedly stimulated PI turnover (3-6 fold) in Swiss, but not Balb/c cells. Insulin (100 ng/ml - 10 ..mu..g/ml) increased total IP to a greater degree in Balb/c cells. Epidermal growth factor (10 ng/ml - 10 ..mu..g/ml) had no effect on PI turnover and fibroblast growth factor (10 ng/ml - 10 ..mu..g/ml) only stimulated at the higher concentrations in Swiss cells. Thrombin (1U/ml - 10 U/ml) produced a 1.5 - 2 fold stimulation in Balb/c cells. Thus, various polypeptide growth factors have differential effects on PI turnover depending on their mitogenic potential and the effector cell type.

  11. Muscarinic receptor-stimulated phosphatidylinositol turnover in the rat corpus striatum: role of muscarinic receptor subtypes and regulation

    SciTech Connect

    Monsma, F.J.

    1987-01-01

    The coupling between the M1 and M2 muscarinic receptor subtypes and phosphatidylinositol (Pl) hydrolysis has been examined in the corpus striatum and cerebral cortex of the rat brain. Receptor binding by the various muscarinic ligands was assessed using a preparation of intact brain cell aggregates, under similar conditions as the assay of Pl hydrolysis. In striatal cell aggregates, (/sup 3/H)-quinuclidinyl benzilate ((/sup 3/H)-QNB) bound to a single class of muscarinic sites with high affinity, inhibition of (/sup 3/H)-QNB binding by muscarinic receptor ligands which exhibit selectivity for subtypes of the muscarinic receptor revealed the presence of both the M1 and M2 subtypes in approximately equal numbers.

  12. Inhibition of Human Class I vs Class III Phosphatidylinositol 3'-Kinases.

    PubMed

    Hassett, Matthew R; Sternberg, Anna R; Roepe, Paul D

    2017-08-22

    Most investigations of phosphatidylinositol 3'-kinase (PI3K) drug inhibition have been via assays based on ADP appearance or ATP consumption (e.g., Liu, Q., et al. ( 2011 ) J. Med. Chem. 54 , 1473 - 1480 ). However, at least some PI3K isoforms show basal ATPase activity in the absence of PI lipid substrate(s), which may complicate quantification of drug potency, isoform specificity of some drugs, and synergy for drug combinations. In this study, we probe the class I vs class III isoform specificity of a selected set of PI3K inhibitors using a simple, inexpensive, semi high-throughput assay that quantifies production of phosphatidylinositol 3'-phosphate (PI3P) from phosphatidylinositol. Results are compared to previous data largely generated using ATPase activity assays. Good agreement between EC50 values computed via ATPase assays vs the reported PI3P formation assay is found for most drugs, but with a few exceptions. Furthermore, for the first time, drug inhibition of class I vs class III enzymes is compared side-by-side with the same assay for the important class I-specific inhibitors GSK2126458 ("Omipalisib") and NVP-BGT226 ("BGT226") currently in clinical development for advanced solid tumors.

  13. Cyclic AMP restores a normal phenotype to sis oncogene transformed cells and inhibits inositol phospholipid turnover

    SciTech Connect

    Murphy, S.K.; Lazarus, A.; Pendergas, M.; Lockwood, A.H.

    1987-05-01

    The sis oncogene encodes the A chain of platelet-derived growth factor (PDGF). NIH3T3 fibroblasts transfected with the cloned sis oncogene display a malignant phenotype and have enhanced turnover of the regulatory phospholipid phosphatidylinositol 4,5 biphosphate (PIP2). They have found that elevation of intracellular cyclic AMP can restore many aspects of normal growth and morphology to sis-transformed cells. Cells rapidly become less refractile, flatten on the substratum, develop actomyosin bundles, and acquire a more tranquil membrane. Growth rate and saturation density are reduced. Cultures become contact-inhibited and, at confluence, assume a normal fibrobastic morphology. The ability to grow in low serum or suspension is lost. Following addition of 8-Br-cAMP, cellular levels of PIP and PIP2 increase to those in untransformed cells. Concurrently, the steady-state levels of inositol phosphates are reduced to normal values. They have found a similar effect of cAMP on inositol phospholipid metabolism in cells transformed by the human H-ras oncogene. These results suggest that cAMP, acting through the cAMP-dependent protein kinase, antagonizes ras and sis oncogene expression by inhibiting polyphosphoinositide turnover. Such action might occur by phosphorylation of the PDGF (sis) receptor or of a ras-stimulated phospholipase C.

  14. Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme.

    PubMed

    Vizitiu, D; Kriste, A G; Campbell, A S; Thatcher, G R

    1996-01-01

    Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.

  15. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

    2013-06-01

    Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

  16. Diosgenin inhibits melanogenesis through the activation of phosphatidylinositol-3-kinase pathway (PI3K) signaling.

    PubMed

    Lee, Jongsung; Jung, Kwangseon; Kim, Yeong Shik; Park, Deokhoon

    2007-06-27

    An increased level of melanin is characteristic of a large number of skin diseases, including acquired hyperpigmentation conditions such as melasma, post inflammatory melanoderma, and solar lentigo. Thus, there is an increasing need for the development of depigmenting agents. In order to evaluate the depigmenting capacity of diosgenin and elucidate its mechanism of action, several experiments were performed in B16 melanoma cells. Melanin content and Western blots for proteins that are involved in melanogenesis were assessed in this study. The melanin content was significantly inhibited by diosgenin. To clarify the mechanism of the depigmenting property of diosgenin, we examined the involvement of diosgenin in the phosphatidylinositol-3-kinase (PI3K) pathway. In this study, diosgenin inhibited the reduction of Akt and GSK 3beta phosphorylation induced by LY294,002, a PI3K inhibitor. In accordance with this result, production levels of MITF (microphthalmia-associated transcription factor) and tyrosinase were increased by diosgenin. These data suggest that diosgenin inhibits melanogenesis through the activation of the PI3K pathway. This suggestion was further confirmed by the fact that the increased production level of melanin by LY294,002 was reduced by diosgenin in B16 melanoma cells. Our study shows that diosgenin inhibits melanogenesis by activating the PI3K pathway, and also suggests that diosgenin may be an effective inhibitor of hyperpigmentation.

  17. A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP2) Inhibition*

    PubMed Central

    Schulte, Bianca; John, Isabel; Simon, Bernd; Brockmann, Christoph; Oelmeier, Stefan A.; Jahraus, Beate; Kirchgessner, Henning; Riplinger, Selina; Carlomagno, Teresa; Wabnitz, Guido H.; Samstag, Yvonne

    2013-01-01

    Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics. PMID:24003227

  18. Creatine inhibits adipogenesis by downregulating insulin-induced activation of the phosphatidylinositol 3-kinase signaling pathway.

    PubMed

    Lee, Nayeon; Kim, Inhee; Park, Soojeong; Han, Dasol; Ha, Soobong; Kwon, Mookwang; Kim, Juwan; Byun, Sung-Hyun; Oh, Wonil; Jeon, Hong Bae; Kweon, Dae-Hyuk; Cho, Jae Youl; Yoon, Keejung

    2015-04-15

    Creatine is a nitrogenous organic acid known to function in adenosine triphosphate (ATP) metabolism. Recent evidence indicates that creatine regulates the differentiation of mesenchymal stem cells (MSCs) in processes such as osteogenesis and myogenesis. In this study, we show that creatine also has a negative regulatory effect on fat cell formation. Creatine inhibits the accumulation of cytoplasmic triglycerides in a dose-dependent manner irrespective of the adipogenic cell models used, including a C3H10T1/2 MSC line, 3T3-L1 preadipocytes, and primary human MSCs. Consistently, a dramatic reduction in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), glucose transporters, 1 and 4 (Glut1, Glut4), and adipocyte markers, aP2 and adipsin, was observed in the presence of creatine. Creatine appears to exert its inhibitory effects on adipogenesis during early differentiation, but not late differentiation, or proliferation stages through inhibition of the PI3K-Akt-PPARγ signaling pathway. In an in vivo model, administration of creatine into mice resulted in body mass increase without fat accumulation. In summary, our results indicate that creatine downregulates adipogenesis through inhibition of phosphatidylinositol 3-kinase (PI3K) activation and imply the potent therapeutic value of creatine in treating obesity and obesity-related metabolic disorders.

  19. Phosphatidylinositol 3-kinase inhibition broadly sensitizes glioblastoma cells to death receptor- and drug-induced apoptosis.

    PubMed

    Opel, Daniela; Westhoff, Mike-Andrew; Bender, Ariane; Braun, Veit; Debatin, Klaus-Michael; Fulda, Simone

    2008-08-01

    The aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway has been reported to correlate with adverse clinical outcome in human glioblastoma in vivo. However, the question of how this survival network can be successfully targeted to restore the sensitivity of glioblastoma to apoptosis induction has not yet been answered. Here, we report that inhibition of PI3K by LY294002 broadly sensitizes wild-type and mutant PTEN glioblastoma cells to both death receptor- and chemotherapy-induced apoptosis, whereas mammalian target of rapamycin (mTOR) inhibition is not sufficient to restore apoptosis sensitivity. LY294002 significantly enhances apoptosis triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), agonistic anti-CD95 antibodies, or several anticancer drugs (i.e., doxorubicin, etoposide, and vincristine) in a highly synergistic manner. In addition, LY294002 cooperates with TRAIL or doxorubicin to suppress colony formation, thus also showing a strong effect on long-term survival. Similarly, genetic knockdown of PI3K subunits p110alpha and/or p110beta by RNA interference (RNAi) primes glioblastoma cells for TRAIL- or doxorubicin-mediated apoptosis. In contrast to PI3K inhibition, pharmacologic or genetic blockade of mTOR by RAD001 (everolimus), rapamycin, or RNAi fails to enhance TRAIL- or doxorubicin-induced apoptosis. Analysis of apoptosis pathways reveals that PI3K inhibition acts in concert with TRAIL or doxorubicin to trigger mitochondrial membrane permeabilization, caspase activation, and caspase-dependent apoptosis, which are abolished by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Most importantly, PI3K inhibition by LY294002 sensitizes primary cultured glioblastoma cells obtained from surgical specimens to TRAIL- or chemotherapy-induced cell death. By showing that PI3K inhibition broadly primes glioblastoma cells for apoptosis, our findings provide the rationale for using PI3K inhibitors in

  20. GABAB receptor stimulation by baclofen and taurine enhances excitatory amino acid induced phosphatidylinositol turnover in neonatal rat cerebellum.

    PubMed

    Smith, S S; Li, J

    1991-10-28

    Excitatory amino acid stimulation of phosphatidylinositol (PI) hydrolysis has been associated with development of the CNS. Normally minimally ineffective in stimulating PI hydrolysis in the neonatal rat cerebellum, N-methyl-D-aspartate (NMDA) increased levels of PI hydrolysis 82.3 +/- 5.5% above basal values in the presence of 1 microM baclofen, a gamma-aminobutyric acidB (GABAB) receptor agonist. This effect was observed at day 7 but not in adult cerebellum. The effect of baclofen could be mimicked by low dose GABA and taurine, actions which were blocked by prior application of a specific GABAB antagonist. Therefore, the ability of NMDA to stimulate PI hydrolysis in neonatal cerebellar tissue may be regulated by the degree of GABAB receptor stimulation.

  1. Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

    SciTech Connect

    Chauhan, A.; Cauhan, V.P.S.; Deshmukh, D.S.; Brokerhoff, H. )

    1989-06-13

    Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}), can also activate PKC in the presence of phosphatidylserine (PS) and Ca{sup 2+} with a K{sub PIP{sub 2}} of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP{sub 2} and DG on PKC. Here, the authors investigate the effect of PIP{sub 2} on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP{sub 2} inhibited specific binding of ({sup 3}H)phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP{sub 2} than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP{sub 2} is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (K{sub d{prime}}) against PIP{sub 2} concentration was linear over a range of 0.01-1 mol % with a K{sub i} of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP{sub 2}. Competition between PIP{sub 2} and phorbol ester could be determined in a liposomal assay system also. These results indicate that PIP{sub 2}, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP{sub 2} is a primary activator of the enzyme.

  2. Phosphatidylinositol 3-kinase inhibition potentiates glucocorticoid response in B-cell acute lymphoblastic leukemia.

    PubMed

    Evangelisti, Cecilia; Cappellini, Alessandra; Oliveira, Mariana; Fragoso, Rita; Barata, João T; Bertaina, Alice; Locatelli, Franco; Simioni, Carolina; Neri, Luca M; Chiarini, Francesca; Lonetti, Annalisa; Buontempo, Francesca; Orsini, Ester; Pession, Andrea; Manzoli, Lucia; Martelli, Alberto Maria; Evangelisti, Camilla

    2017-08-04

    Despite remarkable progress in polychemotherapy protocols, pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains fatal in around 20% of cases. Hence, novel targeted therapies are needed for patients with poor prognosis. Glucocorticoids (GCs) are drugs commonly administrated for B-ALL treatment. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway is frequently observed in B-ALL and contributes to GC-resistance. Here, we analyzed for the first time to our knowledge, the therapeutic potential of pan and isoform-selective PI3K p110 inhibitors, alone or combined with dexamethasone (DEX), in B-ALL leukemia cell lines and patient samples. We found that a pan PI3K p110 inhibitor displayed the most powerful cytotoxic effects in B-ALL cells, by inducing cell cycle arrest and apoptosis. Both a pan PI3K p110 inhibitor and a dual γ/δ PI3K p110 inhibitor sensitized B-ALL cells to DEX by restoring nuclear translocation of the GC receptor and counteracted stroma-induced DEX-resistance. Finally, gene expression analysis documented that, on one hand the combination consisting of a pan PI3K p110 inhibitor and DEX strengthened the DEX-induced up- or down-regulation of several genes involved in apoptosis, while on the other, it rescued the effects of genes that might be involved in GC-resistance. Overall, our findings strongly suggest that PI3K p110 inhibition could be a promising strategy for treating B-ALL patients by improving GC therapeutic effects and/or overcoming GC-resistance. © 2017 Wiley Periodicals, Inc.

  3. Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes.

    PubMed Central

    Hawkins, P T; Michell, R H; Kirk, C J

    1984-01-01

    We have developed methods that yield estimates of the 32P content of each of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, thus extending the information available from studies of the labelling of these lipids in intact cells or membrane preparations. The analyses are undertaken with the deacylated lipids. Assay of the 5-phosphate of phosphatidylinositol 4,5-bisphosphate is achieved by the use, under conditions of first-order kinetics, of a 5-phosphate-specific phosphomonoesterase present in isolated erythrocyte membranes [Downes, Mussat & Michell (1982) Biochem. J. 203, 169-177]. Assay of the 4-phosphate of phosphatidylinositol 4-phosphate and of the total monoester phosphate content (4-phosphate plus 5-phosphate) of phosphatidylinositol 4,5-bisphosphate employs alkaline phosphatase from bovine intestine. The radioactivity of the 1-phosphate is that remaining as organic phosphate after exhaustive alkaline phosphatase treatment. The methodology has been validated by using lipids from human erythrocytes: these contain no 32P in their 1-phosphate. These methods should be of substantial value in studies of the many cells that show rapid hormonal perturbations of phosphatidylinositol 4,5-bisphosphate metabolism. PMID:6326746

  4. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

    PubMed Central

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2010-01-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa α-subunit (p110α) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H2O2 stimulates the expression of p110α in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton. PMID:16204406

  5. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD.

    PubMed

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2006-04-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa alpha-subunit (p110alpha) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H(2)O(2) stimulates the expression of p110alpha in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

  6. Structural Insights into the Inhibition of Actin-Capping Protein by Interactions with Phosphatidic Acid and Phosphatidylinositol (4,5)-Bisphosphate

    PubMed Central

    Pleskot, Roman; Pejchar, Přemysl; Žárský, Viktor; Staiger, Christopher J.; Potocký, Martin

    2012-01-01

    The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP2). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP2/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP–membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro. PMID:23133367

  7. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    PubMed Central

    Li, Jie; Zhang, Chao; Jiang, Hongchuan; Cheng, Jiao

    2015-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro) was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10−7 mol/L), by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF) gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future. PMID:25709476

  8. LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

    SciTech Connect

    Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

    2008-10-01

    Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca{sup 2+} concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

  9. Phosphatidylinositol 4,5-bisphosphate and loss of PLCgamma activity inhibit TRPM channels required for oscillatory Ca2+ signaling.

    PubMed

    Xing, Juan; Strange, Kevin

    2010-02-01

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

  10. Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity

    PubMed Central

    Agarwal, Shivani; Kim, Hyunjin; Chan, Robin B.; Agarwal, Shivangi; Williamson, Rebecca; Cho, Wonhwa; Paolo, Gilbert D.; Satchell, Karla J. F.

    2015-01-01

    Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser–His–Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae. PMID:26498860

  11. Hyperbaric hyperoxia reversibly inhibits erythrocyte phospholipid fatty acid turnover

    NASA Technical Reports Server (NTRS)

    Dise, Craig A.; Clark, James M.; Lambersten, Christian J.; Goodman, David B. P.

    1987-01-01

    The effect of hyperbaric hyperoxia on the acylation of membrane phospholipid was studied by measuring the rates of activation of exogenous tritiated oleic acid to acyl thioester and of transesterification of the thioester into membrane phospholipids in intact human erythrocytes obtained 1 h after an exposure of the subjects to a hyperbaric oxygen atmosphere (3.5 h, 100 pct O2, 3 ATA). Exposure to pure oxygen was found to inhibit both the acylation and transesterification reactions by more than 30 percent, with partial recovery detected 24 h later. On the other hand, no rate changes were observed when isolated membranes from the same batches of cells were used in similar experiments. It is suggested that the decrease in the incorporation of tritiated oleic acid after hyperbaric hyperoxia may reflect an early event in the pathogenesis of oxygen-induced cellular injury and that it may be a useful index for the assessment of the tolerance of tissues to hyperoxia.

  12. Hyperbaric hyperoxia reversibly inhibits erythrocyte phospholipid fatty acid turnover

    NASA Technical Reports Server (NTRS)

    Dise, Craig A.; Clark, James M.; Lambersten, Christian J.; Goodman, David B. P.

    1987-01-01

    The effect of hyperbaric hyperoxia on the acylation of membrane phospholipid was studied by measuring the rates of activation of exogenous tritiated oleic acid to acyl thioester and of transesterification of the thioester into membrane phospholipids in intact human erythrocytes obtained 1 h after an exposure of the subjects to a hyperbaric oxygen atmosphere (3.5 h, 100 pct O2, 3 ATA). Exposure to pure oxygen was found to inhibit both the acylation and transesterification reactions by more than 30 percent, with partial recovery detected 24 h later. On the other hand, no rate changes were observed when isolated membranes from the same batches of cells were used in similar experiments. It is suggested that the decrease in the incorporation of tritiated oleic acid after hyperbaric hyperoxia may reflect an early event in the pathogenesis of oxygen-induced cellular injury and that it may be a useful index for the assessment of the tolerance of tissues to hyperoxia.

  13. Positron emission tomographic monitoring of dual phosphatidylinositol-3-kinase and mTOR inhibition in anaplastic large cell lymphoma

    PubMed Central

    Graf, Nicolas; Li, Zhoulei; Herrmann, Ken; Weh, Daniel; Aichler, Michaela; Slawska, Jolanta; Walch, Axel; Peschel, Christian; Schwaiger, Markus; Buck, Andreas K; Dechow, Tobias; Keller, Ulrich

    2014-01-01

    Background Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[18F]fluoro-D-glucose (FDG) and the thymidine analog, 3′-deoxy-3′-[18F] fluorothymidine (FLT). Methods The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects. Results SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue. Conclusion Dual PI3K/mTOR inhibition using BGT

  14. Positron emission tomographic monitoring of dual phosphatidylinositol-3-kinase and mTOR inhibition in anaplastic large cell lymphoma.

    PubMed

    Graf, Nicolas; Li, Zhoulei; Herrmann, Ken; Weh, Daniel; Aichler, Michaela; Slawska, Jolanta; Walch, Axel; Peschel, Christian; Schwaiger, Markus; Buck, Andreas K; Dechow, Tobias; Keller, Ulrich

    2014-01-01

    Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) and the thymidine analog, 3'-deoxy-3'-[(18)F] fluorothymidine (FLT). The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects. SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue. Dual PI3K/mTOR inhibition using BGT226 is effective in ALK

  15. Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

    SciTech Connect

    Papadopoulos, T.; Pfeifer, U. )

    1987-07-01

    The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with ({sup 14}C)thymidine and ({sup 3}H)leucine. The evaluation of the DNA turnover and additional ({sup 3}H)thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.

  16. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A/sub 2/

    SciTech Connect

    Burch, R.M.; Axelrod, J.

    1987-09-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E/sub 2/ (PGE/sub 2/) synthesis. The EC/sub 50/ values for stimulation of PGE/sub 2/ synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-(..gamma..-thio)triphosphate stimulated PGE/sub 2/ synthesis and InsP formation, and guanosine-5'-(..beta..-thio)diphosphate inhibited both PGE/sub 2/ synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE/sub 2/ synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE/sub 2/ synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE/sub 2/ synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE/sub 2/ synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with (/sup 3/H) choline, the phospholipase A/sub 2/ products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A/sub 2/ and that phospholipase A/sub 2/ is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.

  17. A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

    PubMed Central

    Ozes, Osman Nidai; Akca, Hakan; Mayo, Lindsey D.; Gustin, Jason A.; Maehama, Tomohiko; Dixon, Jack E.; Donner, David B.

    2001-01-01

    Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppessor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN. PMID:11287630

  18. Inhibition of the phosphatidylinositol-specific phospholipase C from Bacillus cereus by a monoclonal antibody binding to a region with sequence similarity to eukaryotic phospholipases.

    PubMed

    Kuppe, A; Hedberg, K K; Volwerk, J J; Griffith, O H

    1990-10-22

    Bacterial phosphatidylinositol-specific phospholipases C (PI-PLC) display similar substrate specificity as their eukaryotic counterparts involved in signal transduction of insulin and Ca2(+)-mobilizing hormones, and are used in the study of the novel glycosylphosphatidylinositol-protein anchors (GPI-anchors). For the investigation of structure-function aspects of the PI-PLC secreted from Bacillus cereus cells, a panel of murine monoclonal antibodies was generated and shown to be specific for the PI-PLC polypeptide in enzyme-linked immunosorbent assays and Western blots. Two of the monoclonals inhibited reactions catalyzed by the bacterial enzyme in vitro: hydrolysis of phosphatidylinositol and the release of bovine erythrocyte acetylcholinesterase from its GPI-anchor. At saturating concentrations of inhibitory antibody only a few percent of the enzyme activity remained. The epitope recognized by one of the inhibitory antibodies, A72-24, was mapped by proteolytic digestion, protein sequencing, and Western blotting of the generated fragments. The data indicate that at least part of the epitope resides within an 8 kDa-stretch of the bacterial PI-PLC (Gln-45 - Lys-122). Essentially the same segment of the bacterial polypeptide has previously been shown to display limited amino acid sequence similarity with several eukaryotic PI-specific phospholipases C (Kuppe, A., Evans, L.M., McMillen, D.A. and Griffith, O.H. (1989) J. Bacteriol. 171, 6077-6083). The results reported here suggest that the conserved peptide of these enzymes may contain functionally important residues.

  19. Inhibition of histamine turnover by 8-OH-DPAT, buspirone and 5-hydroxytryptophan in the mouse and rat brain.

    PubMed

    Oishi, R; Itoh, Y; Saeki, K

    1992-05-01

    The effects of 5-hydroxytryptamine (5-HT) receptor agonists on histamine turnover in mouse and rat brains were examined. The histamine turnover rate was estimated from the accumulation of tele-methylhistamine 90 min after i.p. injection of pargyline (65 mg/kg). In whole mouse brains, the histamine turnover was significantly inhibited by the 5-HT1A agonists, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (greater than 0.5 mg/kg) and buspirone (greater than 2 mg/kg) injected s.c. 10 min before pargyline treatment. 5-hydroxytryptophan (20 mg/kg) also significantly inhibited histamine turnover. Injections of the 5-HT1B agonist m-trifluoromethylphenylpiperazine (10 and 20 mg/kg) or the 5-HT2 agonist (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (1, 2 and 5 mg/kg), however, did not affect histamine turnover. The inhibitory effect of 8-OH-DPAT (1 mg/kg) on histamine turnover was significantly antagonized (by 40%) by pindolol (20 mg/kg) and slightly antagonized (by 29%) by spiperone (10 mg/kg), while methysergide (20 mg/kg) and ketanserin (10 mg/kg) demonstrated no antagonistic effects. 8-OH-DPAT (0.3 and 1 mg/kg) also showed an inhibiting effect on histamine turnover in various regions of rat brains. Although the extent of inhibition was slightly larger in the striatum and cerebral cortex, there was no marked regional difference. These results suggest that histaminergic activity in the brain is regulated by 5-HT1A receptors.

  20. Rapid cytoplasmic turnover of yeast ribosomes in response to rapamycin inhibition of TOR.

    PubMed

    Pestov, Dimitri G; Shcherbik, Natalia

    2012-06-01

    The target of rapamycin (TOR) pathway is the central regulator of cell growth in eukaryotes. Inhibition of TOR by rapamycin elicits changes in translation attributed mainly to altered translation initiation and repression of the synthesis of new ribosomes. Using quantitative analysis of rRNA, we found that the number of existing ribosomes present in a Saccharomyces cerevisiae culture during growth in rich medium rapidly decreases by 40 to 60% when the cells are treated with rapamycin. This process is not appreciably affected by a suppression of autophagy, previously implicated in degradation of ribosomes in eukaryotes upon starvation. Yeast cells deficient in the exosome function or lacking its cytoplasmic Ski cofactors show an abnormal pattern of rRNA degradation, particularly in the large ribosomal subunit, and accumulate rRNA fragments after rapamycin treatment and during diauxic shift. The exosome and Ski proteins are thus important for processing of rRNA decay intermediates, although they are probably not responsible for initiating rRNA decay. The role of cytoplasmic nucleases in rapamycin-induced rRNA degradation suggests mechanistic parallels of this process to nutrient-controlled ribosome turnover in prokaryotes. We propose that ribosome content is regulated dynamically in eukaryotes by TOR through both ribosome synthesis and the cytoplasmic turnover of mature ribosomes.

  1. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  2. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    PubMed Central

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes. PMID:8611143

  3. Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

    SciTech Connect

    McDonald, Gail T.; Sullivan, Richard; Pare, Genevieve C.; Graham, Charles H.

    2010-11-15

    Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G{sub 1} phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}; and knockdown of p27{sup kip1} with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.

  4. Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.

    PubMed Central

    Jhun, B H; Rose, D W; Seely, B L; Rameh, L; Cantley, L; Saltiel, A R; Olefsky, J M

    1994-01-01

    We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction. Images PMID:7935461

  5. Echinacea purpurea root extract inhibits TNF release in response to Pam3Csk4 in a phosphatidylinositol-3-kinase dependent manner.

    PubMed

    Fast, David J; Balles, John A; Scholten, Jeffrey D; Mulder, Timothy; Rana, Jatinder

    2015-10-01

    Polysaccharides derived from Echinacea have historically been shown to be immunostimulatory. We describe in this work however the anti-inflammatory effect of a water extract of Echinacea purpurea roots (EPRW) that inhibited Pam3Csk4 stimulated production of TNFα by human monocytic THP-1 cells. The polyphenols and alkylamides typically found in Echinacea extracts were absent in EPRW suggesting that the anti-inflammatory component(s) was a polysaccharide. This anti-inflammatory activity was shown to be mediated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway as chemical inhibition of PI3K abolished the EPRW anti-inflammatory effect. Demonstration of phosphorylation of Akt and ribosomal S6 proteins, downstream targets of PI3K confirmed EPRW-mediated activation of this pathway. In conclusion, this observation suggests that non-alkylamide/non-polyphenolic phytochemicals from Echinacea may contribute in part to some of the anti-inflammatory therapeutic effects such as reduced severity of symptoms that have been observed in vivo in the treatment of upper respiratory tract infections with Echinacea. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Ellagitannin-rich cloudberry inhibits hepatocyte growth factor induced cell migration and phosphatidylinositol 3-kinase/AKT activation in colon carcinoma cells and tumors in Min mice

    PubMed Central

    Pajari, Anne-Maria; Päivärinta, Essi; Paavolainen, Lassi; Vaara, Elina; Koivumäki, Tuuli; Garg, Ritu; Heiman-Lindh, Anu; Mutanen, Marja; Marjomäki, Varpu; Ridley, Anne J.

    2016-01-01

    Berries have been found to inhibit colon carcinogenesis in animal models, and thus represent a potential source of compounds for prevention and treatment of colorectal cancer. The mechanistic basis for their effects is not well understood. We used human colon carcinoma cells and Min mice to investigate the effects of ellagitannin-rich cloudberry (Rubus chamaemorus) extract on cancer cell migration and underlying cell signaling. Intrinsic and hepatocyte growth factor (HGF) -induced cell motility in human HT29 and HCA7 colon carcinoma cells was assessed carrying out cell scattering and scratch wound healing assays using time-lapse microscopy. Activation of Met, AKT, and ERK in cell lines and tumors of cloudberry-fed Min mice were determined using immunoprecipitation, Western blot and immunohistochemical analyses. Cloudberry extract significantly inhibited particularly HGF-induced cancer cell migration in both cell lines. Cloudberry extract inhibited the Met receptor tyrosine phosphorylation by HGF and strongly suppressed HGF-induced AKT and ERK activation in both HT29 and HCA7 cells. Consistently, cloudberry feeding (10% w/w freeze-dried berries in diet for 10 weeks) reduced the level of active AKT and prevented phosphoMet localization at the edges in tumors of Min mice. These results indicate that cloudberry reduces tumor growth and cancer cell motility by inhibiting Met signaling and consequent activation of phosphatidylinositol 3-kinase/AKT in vitro and in tumors in vivo. As the Met receptor is recognized to be a major target in cancer treatment, our results suggest that dietary phytochemicals may have therapeutic value in reducing cancer progression and metastasis. PMID:27270323

  7. Strategically Timing Inhibition of Phosphatidylinositol 3-Kinase to Maximize Therapeutic Index in Estrogen Receptor Alpha-Positive, PIK3CA-Mutant Breast Cancer.

    PubMed

    Yang, Wei; Hosford, Sarah R; Dillon, Lloye M; Shee, Kevin; Liu, Stephanie C; Bean, Jennifer R; Salphati, Laurent; Pang, Jodie; Zhang, Xiaolin; Nannini, Michelle A; Demidenko, Eugene; Bates, Darcy; Lewis, Lionel D; Marotti, Jonathan D; Eastman, Alan R; Miller, Todd W

    2016-05-01

    Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)-positive breast cancer in combination with antiestrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER(+) breast cancer will provide a rationale for treatment scheduling to maximize therapeutic index. Antiestrogen-sensitive and antiestrogen-resistant ER(+) human breast cancer cell lines and mice bearing PIK3CA-mutant xenografts were treated with the antiestrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than that with single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6 to 9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared with fulvestrant alone. Continuous and metronomic PI3K inhibition elicits robust anticancer effects in ER(+), PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with antiestrogens

  8. Inhibition by atropine of the increased turnover of noradrenaline in the hypothalamus of rats exposed to cold

    PubMed Central

    Simmonds, M. A.

    1971-01-01

    1. Small doses of (-)-[3H] noradrenaline were injected into the lateral cerebral ventricles in rats to label radioactively the endogenous noradrenaline (NA) stores. 2. Intraventricular injection of 25 μg atropine methonitrate at the same time inhibited the increased rate of disappearance of [3H] NA from the hypothalamus at an environmental temperature of 9° C, when compared with the values at 24° C, without impairing temperature regulation. 3. At 32° C, 25 μg atropine methonitrate caused a lethal hyperthermia. A dose of 5 μg was not lethal and did not inhibit the increased rate of disappearance of [3H] NA from the hypothalamus. 4. It is concluded that the pathway which stimulates an increased turnover of NA in the cold contains an atropine sensitive synapse but is not the principal pathway of heat production. The increased turnover of NA in the heat probably does not involve an atropine sensitive synapse. PMID:5091157

  9. Inhibition of focal adhesion kinase induces apoptosis in bladder cancer cells via Src and the phosphatidylinositol 3-kinase/Akt pathway

    PubMed Central

    KONG, DEBO; CHEN, FENG; SIMA, NI

    2015-01-01

    Focal adhesion kinase (FAK) is a 125-kDa, cytosolic, non-receptor, protein tyrosine kinase localized at focal adhesions that can be activated by multiple inputs and in different manners. FAK is implicated in signaling pathways regulating cell movement, invasion, survival, gene expression and cancer stem cell self-renewal. The aim of the present study was to investigate whether FAK plays a role in the apoptosis of bladder cancer cells. The study employed in situ deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and Annexin V labeling flow cytometry. It was found that both the knockdown of FAK and the suppression of FAK phosphorylation were able to induce apoptosis in bladder cancer cells. Caspase-3 was activated during the apoptosis induced by the suppression of FAK phosphorylation. Src was involved in FAK-regulated apoptosis in bladder cancer cells, while the suppression of Src phosphorylation was able to inhibit FAK tyrosine phosphorylation and induce apoptosis. Furthermore, phosphatidylinositol 3-kinase (PI3K)/Akt signaling was inhibited via the suppression of FAK tyrosine phosphorylation. Conversely, the expression of neither the general nor the tyrosine-phosphorylated FAK was regulated by inhibiting PI3K/Akt, which suggested that PI3K/Akt acted downstream of FAK to regulate apoptosis in bladder cancer cells. These findings indicate the presence of a mechanism of apoptosis involving FAK-mediated oncogenic signaling. FAK may function as an important regulator of extracellular signaling-mediated apoptosis in bladder cancer and be used as a novel therapeutic target in the treatment of the condition. PMID:26640543

  10. Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries

    SciTech Connect

    Keating, Aileen F.; Mark, Connie J.; Sen, Nivedita; Sipes, I. Glenn; Hoyer, Patricia B.

    2009-12-01

    4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12 days in vehicle control, VCD (30 muM), or DMBA (1 muM), +- PI3 kinase inhibitor LY294002 (20 muM) or its inactive analog LY303511 (20 muM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P < 0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P < 0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P > 0.05) at any time, but did cause loss (P < 0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P < 0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P < 0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.

  11. Autophagy inhibition enhances colorectal cancer apoptosis induced by dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235

    PubMed Central

    YANG, XIAOYU; NIU, BINGXUAN; WANG, LIBO; CHEN, MEILING; KANG, XIAOCHUN; WANG, LUONAN; JI, YINGHUA; ZHONG, JIATENG

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway performs a central role in tumorigenesis and is constitutively activated in many malignancies. As a novel dual PI3K/mTOR inhibitor currently undergoing evaluation in a phase I/II clinical trial, NVP-BEZ235 indicates a significant antitumor efficacy in diverse solid tumors, including colorectal cancer (CRC). Autophagy is a catabolic process that maintains cellular homeostasis and reduces diverse stresses through lysosomal recycling of the unnecessary and damaged cell components. This process is also observed to antagonize the antitumor efficacy of PI3K/mTOR inhibitor agents such as NVP-BEZ235, via apoptosis inhibition. In the present study, we investigated anti-proliferative and apoptosis-inducing ability of NVP-BEZ235 in SW480 cells and the crosstalk between autophagy and apoptosis in SW480 cells treated with NVP-BEZ235 in combination with an autophagy inhibitor. The results revealed that, NVP-BEZ235 effectively inhibit the growth of SW480 cells by targeting the PI3K/mTOR signaling pathway and induced apoptosis. The inhibition of autophagy with 3-methyladenine or chloroquine inhibitors in combination with NVP-BEZ235 in SW480 cells enhanced the apoptotic rate as componets to NVP-BEZ235 alone. In conclusion, the findings provide a rationale for chemotherapy targeting the PI3K/mTOR signaling pathway presenting a potential therapeutic strategy to enhance the efficacy of dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with an autophagy inhibitor in CRC treatment and treatment of other tumors. PMID:27347108

  12. miR-362-5p inhibits proliferation and migration of neuroblastoma cells by targeting phosphatidylinositol 3-kinase-C2β.

    PubMed

    Wu, Kai; Yang, Liucheng; Chen, Jianfeng; Zhao, Haijun; Wang, Jianjun; Xu, Shuai; Huang, Zonghai

    2015-07-08

    miR-362-5p is down-regulated in high-risk neuroblastoma and can function as a tumor suppressor. However, its role remains poorly understood. We show that miR-362-5p is down-regulated in metastatic neuroblastoma compared with primary neuroblastoma. Overexpression of miR-362-5p inhibits cell proliferation, migration and invasion of neuroblastoma cells in vitro and suppresses tumor growth of neuroblastoma in vivo. Phosphatidylinositol 3-kinase (PI3K)-C2β is a target of miR-362-5p. Knockdown of PI3K-C2β by siRNA had a similar effect to overexpression of miR-362-5p on SH-SY5Y cells. Overexpression of PI3K-C2β partially reversed tumor-suppressive effects of miR-362-5p. We suggest that miR-362-5p suppresses neuroblastoma cell growth and motility, partially by targeting PI3K-C2β.

  13. Type II cGMP-dependent protein kinase inhibits epidermal growth factor-induced phosphatidylinositol-3-kinase/Akt signal transduction in gastric cancer cells.

    PubMed

    Wu, Min; Chen, Yongchang; Jiang, Lu; Li, Yueying; Lan, Ting; Wang, Ying; Qian, Hai

    2013-12-01

    Our previous study revealed that Type II cGMP-dependent protein kinase (PKG II) inhibits epidermal growth factor (EGF)-induced MAPK/ERK and MAPK/JNK-mediated signal transduction through the inhibition of the phosphorylation/activation of the EGF receptor (EGFR). As EGFR also mediates several other signal transduction pathways besides MAPK-mediated pathways, the present study was designed to investigate whether PKG II was able to inhibit EGF/EGFR-induced phosphatidylinositol-3-kinase (PI3K)/Akt-mediated signal transduction. The AGS human gastric cancer cell line was infected with adenoviral constructs encoding a cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II, and treated with 8-pCPT-cGMP to activate the enzyme. Western blotting was used to detect the phosphorylation/activation of the key components of the signal transduction pathway, including EGFR, PI3K, Akt, mTOR and NF-κB. The levels of apoptosis-related proteins, including Bax, Bcl-2, caspase 9 and DNA fragment factor (DFF), were also determined by western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining was used to detect the apoptosis of the AGS cells. The results revealed that EGF treatment increased the phosphorylation (activation) of EGFR, PI3K, Akt and mTOR, and increased the nuclear localization (activation) of NF-κB. EGF treatment also reduced the apoptosis of the AGS cells and increased the expression of the anti-apoptotic protein, Bcl-2, but had no effect on the expression of the pro-apoptotic protein, Bax, and did not alter the levels of caspase 9 and DFF. Increasing the PKG II activity of AGS cells by infecting them with Ad-PKG II and stimulating them with 8-pCPT-cGMP inhibited the EGF-induced activation of EGFR, PI3K, Akt, mTOR and NF-κB; caused an increase in caspase 9 breakdown (activation) and DFF levels; and reversed the anti-apoptotic effect of EGF. The results suggest that PKG II may also inhibit EGF-induced signal transduction of PI3

  14. Blueberry Phytochemicals Inhibit Growth and Metastatic Potential of MDA-MB-231 Breast Cancer Cells Through Modulation of the Phosphatidylinositol 3-Kinase Pathway

    PubMed Central

    Adams, Lynn S.; Phung, Sheryl; Yee, Natalie; Seeram, Navindra P.; Li, Liya; Chen, Shiuan

    2010-01-01

    Dietary phytochemicals are known to exhibit a variety of anti-carcinogenic properties. This study investigated the chemopreventive activity of blueberry extract in triple negative breast cancer cell lines in vitro and in vivo. Blueberry decreased cell proliferation in HCC38, HCC1937 and MDA-MB-231 cells with no effect on the non-tumorigenic MCF-10A cell line. Decreased metastatic potential of MDA-MB-231 cells by blueberry was shown through inhibition of cell motility using wound healing assays and migration through a PET membrane. Blueberry treatment decreased the activity of matrix metalloproteinase 9 and the secretion of urokinase-type plasminogen activator while increasing tissue inhibitor of metalloproteinase-1 and plasminogen activator inhibitor-1 secretion in MDA-MB-231 conditioned medium as shown by western blotting. Cell signaling pathways that control the expression/activation of these processes were investigated via western blotting and reporter gene assay. Treatment with blueberry decreased phosphatidylinositol 3-kinase (PI3K)/AKT and nuclear factor kappa-B (NFκB) activation in MDA-MB-231 cells where protein kinase C (PKC) and extracellular regulated kinase (ERK) were not affected. In vivo, the efficacy of blueberry to inhibit triple negative breast tumor growth was evaluated using the MDA-MB-231 xenograft model. Tumor weight and proliferation (Ki-67 expression) were decreased in blueberry treated mice, where apoptosis (caspase-3 expression) was increased compared to controls. Immunohistochemical analysis of tumors from blueberry-fed mice showed decreased activation of AKT and p65 NFκB signaling proteins with no effect on the phosphorylation of ERK. These data illustrate the inhibitory effect of blueberry phytochemicals on the growth and metastatic potential of MDA-MB-231 cells through modulation of the PI3K/AKT/NFκB pathway. PMID:20388778

  15. Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4(+) T Cell Proliferation.

    PubMed

    Celada, Lindsay J; Rotsinger, Joseph E; Young, Anjuli; Shaginurova, Guzel; Shelton, Debresha; Hawkins, Charlene; Drake, Wonder P

    2017-01-01

    Patients with progressive sarcoidosis exhibit increased expression of programmed death-1 (PD-1) receptor on their CD4(+) T cells. Up-regulation of this marker of T cell exhaustion is associated with a reduction in the proliferative response to T cell receptor (TCR) stimulation, a defect that is reversed by PD-1 pathway blockade. Genome-wide association studies and microarray analyses have correlated signaling downstream from the TCR with sarcoidosis disease severity, but the mechanism is not yet known. Reduced phosphatidylinositol 3-kinase (PI3K)/AKT expression inhibits proliferation by inhibiting cell cycle progression. To test the hypothesis that PD-1 expression attenuates TCR-dependent activation of PI3K/AKT activity in progressive systemic sarcoidosis, we analyzed PI3K/AKT/mechanistic target of rapamycin (mTOR) expression at baseline and after PD-1 pathway blockade in CD4(+) T cells isolated from patients with sarcoidosis and healthy control subjects. We confirmed an increased percentage of PD-1(+) CD4(+) T cells and reduced proliferative capacity in patients with sarcoidosis compared with healthy control subjects (P < 0.001). There was a negative correlation with PD-1 expression and proliferative capacity (r = -0.70, P < 0.001). Expression of key mediators of cell cycle progression, including PI3K and AKT, were significantly decreased. Gene and protein expression levels reverted to healthy control levels after PD-1 pathway blockade. Reduction in sarcoidosis CD4(+) T cell proliferative capacity is secondary to altered expression of key mediators of cell cycle progression, including the PI3K/AKT/mTOR pathway, via PD-1 up-regulation. This supports the concept that PD-1 up-regulation drives the immunologic deficits associated with sarcoidosis severity by inducing signaling aberrancies in key mediators of cell cycle progression.

  16. Phosphatidylinositol 4-kinase III beta is essential for replication of human rhinovirus and its inhibition causes a lethal phenotype in vivo.

    PubMed

    Spickler, Catherine; Lippens, Julie; Laberge, Marie-Kristine; Desmeules, Sophie; Bellavance, Édith; Garneau, Michel; Guo, Tim; Hucke, Oliver; Leyssen, Pieter; Neyts, Johan; Vaillancourt, Fréderic H; Décor, Anne; O'Meara, Jeff; Franti, Michael; Gauthier, Annick

    2013-07-01

    Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIβ). A good correlation between PI4KIIIβ activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIβ inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIβ inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIβ were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIβ is deleterious.

  17. Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

    PubMed

    Göckeritz, Elisa; Kerwien, Susan; Baumann, Michael; Wigger, Marion; Vondey, Verena; Neumann, Lars; Landwehr, Thomas; Wendtner, Clemens M; Klein, Christian; Liu, Ningshu; Hallek, Michael; Frenzel, Lukas P; Krause, Günter

    2015-11-01

    Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

  18. 5-Stabilized phosphatidylinositol 3,4,5-trisphosphate analogues bind Grp1 PH, inhibit phosphoinositide phosphatases, and block neutrophil migration.

    PubMed

    Zhang, Honglu; He, Ju; Kutateladze, Tatiana G; Sakai, Takahiro; Sasaki, Takehiko; Markadieu, Nicolas; Erneux, Christophe; Prestwich, Glenn D

    2010-02-15

    Metabolically stabilized analogues of PtdIns(3,4,5)P3 have shown long-lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5-phosphatase-resistant analogues of PtdIns(3,4,5)P3, the 5-methylene phosphonate (MP) and 5-phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P3 analogues in four contexts. First, the relative binding affinities of the 3-MP, 3-PT, 5-MP, 5-PT, and 3,4,5-PT3 analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P3. Third, exogenously delivered analogues severely impair complement factor C5a-mediated polarization and migration of murine neutrophils. Finally, the new analogues show long-lived agonist activity in mimicking insulin action in sodium transport in A6 cells.

  19. Semaphorin 3B inhibits the phosphatidylinositol 3-kinase/Akt pathway through neuropilin-1 in lung and breast cancer cells.

    PubMed

    Castro-Rivera, Emely; Ran, Sophia; Brekken, Rolf A; Minna, John D

    2008-10-15

    Semaphorin 3B (SEMA3B), located at 3p21.3, is a secreted member of the semaphorin family important in axonal guidance. SEMA3B undergoes allele and expression loss in lung and breast cancer and can function as a tumor suppressor. Previously, we found that SEMA3B induces apoptosis in tumor cells either by reexpression or when applied as a soluble ligand. SEMA3B-induced apoptosis was mediated, in part, by blocking vascular endothelial growth factor autocrine activity in tumor cells. In the current study, treatment of lung and breast cancer cells with picomolar concentrations of soluble SEMA3B inhibited their growth; induced apoptosis; and was associated with decreased Akt phosphorylation, increase in cytochrome c release and caspase-3 cleavage, as well as increased phosphorylation of several proapoptotic proteins, including glycogen synthase kinase-3beta, FKHR, and MDM-2. Lung and breast cancer lines resistant to SEMA3B did not show these signaling changes and a tumor-derived missense SEMA3B mutant was inactive in this regard, providing specificity. SEMA3B-mediated inhibition of proliferation and induction of apoptosis in cancer cells were blocked by expressing a constitutively active Akt mutant and are linked to tumor cell expression of neuropilin-1 (Np-1). SEMA3B-insensitive Np-1-negative tumor cells acquired sensitivity to SEMA3B after forced expression of Np-1, whereas SEMA3B-sensitive Np-1-positive tumor cells lost sensitivity to SEMA3B after knockdown of Np-1 by small interfering RNA. We conclude that SEMA3B is a potential tumor suppressor that induces apoptosis in SEMA3B-inactivated tumor cells through the Np-1 receptor by inactivating the Akt signaling pathway. CA118384

  20. Semaphorin 3B Inhibits the Phosphatidylinositol 3-Kinase/Akt Pathway through Neuropilin-1 in Lung and Breast Cancer Cells

    PubMed Central

    Castro-Rivera, Emely; Ran, Sophia; Brekken, Rolf A.; Minna, John D.

    2009-01-01

    Semaphorin 3B (SEMA3B), located at 3p21.3, is a secreted member of the semaphorin family important in axonal guidance. SEMA3B undergoes allele and expression loss in lung and breast cancer and can function as a tumor suppressor. Previously, we found that SEMA3B induces apoptosis in tumor cells either by reexpression or when applied as a soluble ligand. SEMA3B-induced apoptosis was mediated, in part, by blocking vascular endothelial growth factor autocrine activity in tumor cells. In the current study, treatment of lung and breast cancer cells with picomolar concentrations of soluble SEMA3B inhibited their growth; induced apoptosis; and was associated with decreased Akt phosphorylation, increase in cytochrome c release and caspase-3 cleavage, as well as increased phosphorylation of several proapoptotic proteins, including glycogen synthase kinase-3β, FKHR, and MDM-2. Lung and breast cancer lines resistant to SEMA3B did not showthese signaling changes and a tumor-derived missense SEMA3B mutant was inactive in this regard, providing specificity. SEMA3B-mediated inhibition of proliferation and induction of apoptosis in cancer cells were blocked by expressing a constitutively active Akt mutant and are linked to tumor cell expression of neuropilin-1 (Np-1). SEMA3B-insensitive Np-1–negative tumor cells acquired sensitivity to SEMA3B after forced expression of Np-1, whereas SEMA3B-sensitive Np-1–positive tumor cells lost sensitivity to SEMA3B after knockdown of Np-1 by small interfering RNA. We conclude that SEMA3B is a potential tumor suppressor that induces apoptosis in SEMA3B-inactivated tumor cells through the Np-1 receptor by inactivating the Akt signaling pathway. PMID:18922901

  1. Inhibition of Phosphatidylinositol 3-Kinase/Akt Signaling Suppresses Tumor Cell Proliferation and Neuroendocrine Marker Expression in GI Carcinoid Tumors

    PubMed Central

    Pitt, Susan C.; Chen, Herbert; Kunnimalaiyaan, Muthusamy

    2010-01-01

    Background Over-activation of PI3K/Akt signaling facilitates tumor proliferation in several cancers. We have shown that various signal transduction pathways promote tumorigenesis in carcinoid tumors, which exhibit endogenously high levels of active, phosphorylated Akt. Therefore, we hypothesized that inhibition of the PI3K/Akt pathway would suppress carcinoid tumor cell growth and neuroendocrine (NE) marker production. Methods Human carcinoid BON cells were treated in vitro with LY294002, a PI3 kinase inhibitor, or transfected with Akt1 siRNA. Tumor cell proliferation was measured by MTT for six days. The effect of LY294002 or Akt1 siRNA treatment was assessed by western analysis. We examined the levels of phosphorylated Akt, total Akt, Akt1, and the NE markers human achaete-scute homolog1 (ASCL1) and chromogranin A (CgA). Results Treatment of BON cells with LY294002 reduced tumor cell proliferation (76%) in a dose-dependent manner. Growth also decreased in Akt1 siRNA transfected cells (29%). Levels of active, phosphorylated Akt and the NE tumor markers, ASCL1 and CgA, were diminished with both LY294002 and Akt1 siRNA treatments proportional to the degree of Akt inhibition. Total Akt, Akt2, and Akt3 levels were unaffected by these experiments. Conclusions These data indicate that PI3K/Akt signaling performs a critical role in human carcinoid tumor cell survival and NE hormone generation. Furthermore, the development of novel therapeutics targeting Akt1 or components of the PI3K/Akt pathway may enhance the management of carcinoid disease. Synopsis Carcinoid tumor cells were treated with a PI3K inhibitor, LY294002, and Akt1 siRNA to delineate the role of PI3K/Akt signaling in carcinoids. The effects of treatment on cellular proliferation and neuroendocrine marker expression were observed. PMID:19588205

  2. PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

    PubMed

    Desai, S; Pillai, P; Win-Piazza, H; Acevedo-Duncan, M

    2011-06-01

    The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

  3. Dual inhibition of class IA phosphatidylinositol 3-kinase and mammalian target of rapamycin as a new therapeutic option for T-cell acute lymphoblastic leukemia.

    PubMed

    Chiarini, Francesca; Falà, Federica; Tazzari, Pier Luigi; Ricci, Francesca; Astolfi, Annalisa; Pession, Andrea; Pagliaro, Pasqualepaolo; McCubrey, James A; Martelli, Alberto M

    2009-04-15

    Recent investigations have documented that constitutively activated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it strongly influences growth and survival. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. However, our knowledge of PI3K/Akt/mTOR signaling in T-ALL is limited and it is not clear whether it could be an effective target for innovative therapeutic strategies. Here, we have analyzed the therapeutic potential of the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class, on both T-ALL cell lines and patient samples, which displayed constitutive activation of PI3K/Akt/mTOR signaling. PI-103 inhibited the growth of T-ALL cells, including 170-kDa P-glycoprotein overexpressing cells. PI-103 cytotoxicity was independent of p53 gene status. PI-103 was more potent than inhibitors that are selective only for PI3K (Wortmannin, LY294002) or for mTOR (rapamycin). PI-103 induced G(0)-G(1) phase cell cycle arrest and apoptosis, which was characterized by activation of caspase-3 and caspase-9. PI-103 caused Akt dephosphorylation, accompanied by dephosphorylation of the Akt downstream target, glycogen synthase kinase-3beta. Also, mTOR downstream targets were dephosphorylated in response to PI-103, including p70S6 kinase, ribosomal S6 protein, and 4E-BP1. PI-103 strongly synergized with vincristine. These findings indicate that multitargeted therapy toward PI3K and mTOR alone or with existing drugs may serve as an efficient treatment toward T-ALL cells, which require up-regulation of PI3K/Akt/mTOR signaling for their survival and growth.

  4. Phosphatidylinositol 4,5-bisphosphate degradation inhibits the Na+/bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus oocytes

    PubMed Central

    Thornell, Ian M; Bevensee, Mark O

    2015-01-01

    The electrogenic Na+/bicarbonate cotransporter (NBCe1) of the Slc4 gene family is a powerful regulator of intracellular pH (pHi) and extracellular pH (pHo), and contributes to solute reabsorption and secretion in many epithelia. Using Xenopus laevis oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) directly stimulates NBCe1-A in an excised macropatch, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate (InsP3)/Ca2+. In the current study, we used the two-electrode voltage-clamp technique alone or in combination with pH/voltage-sensitive microelectrodes or confocal fluorescence imaging of plasma membrane PIP2 to characterize the PIP2 sensitivity of NBCe1-B and -C in whole oocytes by co-expressing a voltage-sensitive phosphatase (VSP) that decreases PIP2 and bypasses the InsP3/Ca2+ pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1-B/C current, consistent with an initial rapid depolarization-induced NBCe1 activation, and then a subsequent slower VSP-mediated NBCe1 inhibition. Upon repolarization, the NBCe1 current decreased, and then slowly recovered with an exponential time course that paralleled PIP2 resynthesis as measured with a PIP2-sensitive fluorophore and confocal imaging. A subthreshold depolarization that minimally activated VSP caused a more sustained increase in NBCe1 current, and did not lead to an exponential current recovery following repolarization. Similar results were obtained with oocytes expressing a catalytically dead VSP mutant at all depolarized potentials. Depleting endoplasmic reticulum Ca2+ did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP3/Ca2+ were not responsible. This study demonstrates for the first time that depleting PIP2 per se inhibits NBCe1 activity. The data in conjunction with

  5. The influence of vasopressin and related peptides on glycogen phosphorylase activity and phosphatidylinositol metabolism in hepatocytes.

    PubMed Central

    Kirk, C J; Rodrigues, L M; Hems, D A

    1979-01-01

    The relative abilities of seven vasopressin-like peptides to activate hepatic glycogen phosphorylase and stimulate phosphate incorporation into phosphatidylinositol were compared. Although the individual peptides differed in their potencies, the concentrations required to stimulate phosphatidylinositol metabolism were always greater (about 10 times) than those needed to activate phosphorylase. The molecular specificity of the hepatic vasopressin receptor and the role of vasopressin-stimulated phosphatidylinositol turnover are discussed. PMID:444224

  6. Localization of phosphatidylinositol signaling components in rat taste cells: Role in bitter taste transduction

    SciTech Connect

    Hwang, P.M.; Verma, A.; Bredt, D.S.; Snyder, S.H. )

    1990-10-01

    To assess the role of phosphatidylinositol turnover in taste transduction we have visualized, in rat tongue, ATP-dependent endoplasmic reticular accumulation of {sup 45}Ca{sup 2+}, inositol 1,4,5-trisphosphate receptor binding sites, and phosphatidylinositol turnover monitored by autoradiography of ({sup 3}H)cytidine diphosphate diacylglycerol formed from ({sup 3}H)cytidine. Accumulated {sup 45}Ca{sup 2+}, inositol 1,4,5-trisphosphate receptors, and phosphatidylinositol turnover are selectively localized to apical areas of the taste buds of circumvallate papillae, which are associated with bitter taste. Further evidence for a role of phosphatidylinositol turnover in bitter taste is our observation of a rapid, selective increase in mass levels of inositol 1,4,5-trisphosphate elicited by low concentrations of denatonium, a potently bitter tastant.

  7. Inhibition of glucose turnover by 3-bromopyruvate counteracts pancreatic cancer stem cell features and sensitizes cells to gemcitabine.

    PubMed

    Isayev, Orkhan; Rausch, Vanessa; Bauer, Nathalie; Liu, Li; Fan, Pei; Zhang, Yiyao; Gladkich, Jury; Nwaeburu, Clifford C; Mattern, Jürgen; Mollenhauer, Martin; Rückert, Felix; Zach, Sebastian; Haberkorn, Uwe; Gross, Wolfgang; Schönsiegel, Frank; Bazhin, Alexandr V; Herr, Ingrid

    2014-07-15

    According to the cancer stem cell (CSC) hypothesis, the aggressive growth and early metastasis of pancreatic ductal adenocarcinoma (PDA) is due to the activity of CSCs, which are not targeted by current therapies. Otto Warburg suggested that the growth of cancer cells is driven by a high glucose metabolism. Here, we investigated whether glycolysis inhibition targets CSCs and thus may enhance therapeutic efficacy. Four established and 3 primary PDA cell lines, non-malignant cells, and 3 patient-tumor-derived CSC-enriched spheroidal cultures were analyzed by glucose turnover measurements, MTT and ATP assays, flow cytometry of ALDH1 activity and annexin positivity, colony and spheroid formation, western blotting, electrophoretic mobility shift assay, xenotransplantation, and immunohistochemistry. The effect of siRNA-mediated inhibition of LDH-A and LDH-B was also investigated. The PDA cells exhibited a high glucose metabolism, and glucose withdrawal or LDH inhibition by siRNA prevented growth and colony formation. Treatment with the anti-glycolytic agent 3-bromopyruvate almost completely blocked cell viability, self-renewal potential, NF-κB binding activity, and stem cell-related signaling and reverted gemcitabine resistance. 3-bromopyruvate was less effective in weakly malignant PDA cells and did not affect non-malignant cells, predicting minimal side effects. 3-bromopyruvate inhibited in vivo tumor engraftment and growth on chicken eggs and mice and enhanced the efficacy of gemcitabine by influencing the expression of markers of proliferation, apoptosis, self-renewal, and metastasis. Most importantly, primary CSC-enriched spheroidal cultures were eliminated by 3-bromopyruvate. These findings propose that CSCs may be specifically dependent on a high glucose turnover and suggest 3-bromopyruvate for therapeutic intervention.

  8. CAL-101, a p110δ selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability

    PubMed Central

    Meadows, Sarah A.; Herman, Sarah E. M.; Kashishian, Adam; Steiner, Bart; Johnson, Amy J.; Byrd, John C.; Tyner, Jeffrey W.; Loriaux, Marc M.; Deininger, Mike; Druker, Brian J.; Puri, Kamal D.; Ulrich, Roger G.; Giese, Neill A.

    2011-01-01

    Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ as a treatment approach for patients with B-cell malignancies. We thus identified 5-fluoro-3-phenyl-2-[(S)-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (CAL-101), a highly selective and potent p110δ small molecule inhibitor (half-maximal effective concentration [EC50] = 8nM). Using tumor cell lines and primary patient samples representing multiple B-cell malignancies, we have demonstrated that constitutive phosphatidylinositol-3-kinase pathway activation is p110δ-dependent. CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decreased phosphorylation of Akt and other downstream effectors, an increase in poly(ADP-ribose) polymerase and caspase cleavage and an induction of apoptosis. These effects have been observed across a broad range of immature and mature B-cell malignancies, thereby providing a rationale for the ongoing clinical evaluation of CAL-101. PMID:20959606

  9. CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability.

    PubMed

    Lannutti, Brian J; Meadows, Sarah A; Herman, Sarah E M; Kashishian, Adam; Steiner, Bart; Johnson, Amy J; Byrd, John C; Tyner, Jeffrey W; Loriaux, Marc M; Deininger, Mike; Druker, Brian J; Puri, Kamal D; Ulrich, Roger G; Giese, Neill A

    2011-01-13

    Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ as a treatment approach for patients with B-cell malignancies. We thus identified 5-fluoro-3-phenyl-2-[(S)-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (CAL-101), a highly selective and potent p110δ small molecule inhibitor (half-maximal effective concentration [EC(50)] = 8nM). Using tumor cell lines and primary patient samples representing multiple B-cell malignancies, we have demonstrated that constitutive phosphatidylinositol-3-kinase pathway activation is p110δ-dependent. CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decreased phosphorylation of Akt and other downstream effectors, an increase in poly(ADP-ribose) polymerase and caspase cleavage and an induction of apoptosis. These effects have been observed across a broad range of immature and mature B-cell malignancies, thereby providing a rationale for the ongoing clinical evaluation of CAL-101.

  10. Follicle-stimulating hormone-induced aromatase in immature rat Sertoli cells requires an active phosphatidylinositol 3-kinase pathway and is inhibited via the mitogen-activated protein kinase signaling pathway.

    PubMed

    McDonald, Claudia A; Millena, Ana C; Reddy, Sheila; Finlay, Sheila; Vizcarra, Jorge; Khan, Shafiq A; Davis, John S

    2006-03-01

    Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and

  11. The psychoactive compound of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC) inhibits the human trophoblast cell turnover.

    PubMed

    Costa, M A; Fonseca, B M; Marques, F; Teixeira, N A; Correia-da-Silva, G

    2015-08-06

    The noxious effects of cannabis consumption for fertility and pregnancy outcome are recognized for years. Its consumption during gestation is associated with alterations in foetal growth, low birth weight and preterm labor. The main psychoactive molecule of cannabis, Δ(9)-tetrahydrocannabinol (THC) impairs the production of reproductive hormones and is also able to cross the placenta barrier. However, its effect on the main placental cells, the trophoblasts, are unknown. Actually, the role of THC in cell survival/death of primary human cytotrophoblasts (CTs) and syncytiotrophoblasts (STs) and in the syncytialization process remains to be explored. Here, we show that THC has a dual effect, enhancing MTT metabolism at low concentrations, whereas higher doses decreased cell viability, on both trophoblast phenotypes, though the effects on STs were more evident. THC also diminished the generation of oxidative and nitrative stress and the oxidized form of glutathione, whereas the reduced form of this tripeptide was increased, suggesting that THC prevents ST cell death due to an antioxidant effect. Moreover, this compound enhanced the mitochondrial function of STs, as observed by the increased MTT metabolism and intracellular ATP levels. These effects were independent of cannabinoid receptors activation. Besides, THC impaired CT differentiation into STs, since it decreased the expression of biochemical and morphological biomarkers of syncytialization, through a cannabinoid receptor-dependent mechanism. Together, these results suggest that THC interferes with trophoblast turnover, preventing trophoblast cell death and differentiation, and contribute to disclose the cellular mechanisms that lead to pregnancy complications in women that consume cannabis-derived drugs during gestation.

  12. MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition

    PubMed Central

    Dickson, Mark A.; Klein, Mary E.; O'Connor, Rachael; Wilder, Fatima O.; Socci, Nicholas D.; Tap, William D.; Schwartz, Gary K.; Singer, Samuel; Crago, Aimee M.; Koff, Andrew

    2015-01-01

    CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy. PMID:25803170

  13. Cysteamine inhibition of (/sup 15/N)-glycine turnover in cystinosis and of glycine cleavage system in vitro

    SciTech Connect

    Yudkoff, M.; Nissim, I.; Schneider, A.; Segal, S.

    1981-01-01

    In order to clarify the hyperglycinemic effect of cysteamine treatment in children with nephropathic cystinosis, we measured (/sup 15/N)-glycine turnover in three affected patients. Administration of cysteamine lowered the glycine flux and the glycine metabolic clearance rate but did not alter the glycine pool size. Formation of (/sup 15/N)-serine from (/sup 15/N)-glycine was lower in untreated patients than in control subjects and was reduced still further by cysteamine. Studies in vitro with isolated rat liver mitochondria and acetone extracts of mitochondria indicated that even low cysteamine concentrations (0.1 mM) inhibited the glycine cleavage system in both the direction of glycine oxidation and glycine synthesis. Cysteamine was a more potent inhibitor of the glycine cleavage system than any other sulfhydryl containing compound. Although no ill effects of cysteamine treatment were immediately apparent, patients receiving cysteamine should be monitored carefully for the appearance of any neurologic symptoms which might be referable to inhibition of the glycine cleavage system.

  14. Inhibition by oxytocin of methamphetamine-induced hyperactivity related to dopamine turnover in the mesolimbic region in mice.

    PubMed

    Qi, Jia; Yang, Jing-Yu; Song, Ming; Li, Yan; Wang, Fang; Wu, Chun-Fu

    2008-02-01

    Accumulated data have shown the neuroactive properties of oxytocin (OT), a neurohypophyseal neuropeptide, and its capability of reducing the abuse potential of drugs. The present study investigated the effect of OT on methamphetamine (MAP)-induced hyperactivity in mice and its possible mechanism of action. Locomotor activity was measured after administered with MAP using an infrared sensor. High-performance liquid chromatography with electrochemical detection (HPLC-ECD) was used to detect the content of monoamines and their metabolites in the striatum and accumbens and prefrontal cortex in mice after the behavioral test. OT (0.1, 0.5, and 2.5 microg/mouse, i.c.v.) had no effect on locomotor activity in naïve mice, but inhibited, in a dose-dependent manner, the hyperactivity induced by acute administration of MAP. Atosiban (Ato) (2.0 microg/mouse, i.c.v.), the selective inhibitor of OT receptor, attenuated the inhibitory effect of OT on MAP. A marked reduction of the ratios of 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) to dopamine (DA) was observed in the striatum and accumbens of mice after acute administration of MAP. OT (2.5 microg, i.c.v.) significantly inhibited the reduction of DOPAC/DA and HVA/DA ratios. However, Ato decreased the ratio of DOPAC/DA significantly in mice compared with OT (2.5 microg) in combination with MAP. There was no significant change in serotonin (5-HT) metabolism in mice after a single administration of MAP. These results suggested that OT inhibited the MAP-induced hyperactivity by altering the DA turnover in the mesolimbic region of mice.

  15. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion

    PubMed Central

    Liu, Kai; Jian, Youli; Sun, Xiaojuan; Yang, Chengkui; Gao, Zhiyang; Zhang, Zhili; Liu, Xuezhao; Li, Yang; Xu, Jing; Jing, Yudong; Mitani, Shohei; He, Sudan

    2016-01-01

    Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion. PMID:26783301

  16. Inhibition of neuronal mitochondrial complex I or lysosomal glucocerebrosidase is associated with increased dopamine and serotonin turnover.

    PubMed

    de la Fuente, Carmen; Burke, Derek G; Eaton, Simon; Heales, Simon J R

    2017-02-24

    Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic and serotoninergic signalling. A number of pathogenic mechanisms have been implicated including loss of mitochondrial function at the level of complex I, and lysosomal metabolism at the level of lysosomal glucocerebrosidase (GBA1). In order to investigate further the potential involvement of complex I and GBA1 in PD, we assessed the impact of loss of respective enzyme activities upon dopamine and serotonin turnover. Using SH-SY5Y cells, complex I deficiency was modelled by using rotenone whilst GBA1 deficiency was modelled by the use of conduritol B epoxide (CBE). Dopamine, its principal metabolites, and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the extracellular medium were quantified by HPLC. Inhibition of complex I significantly increased extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-HIAA. Comparable results were observed with CBE. These results suggest increased monoamine oxidase activity and provide evidence for involvement of impaired complex I or GBA1 activity in the dopamine/serotonin deficiency seen in PD. Use of extracellular media may also permit relatively rapid assessment of dopamine/serotonin metabolism and permit screening of novel therapeutic agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Enhanced phosphodiesteratic breakdown and turnover of phosphoinositides during reperfusion of ischemic rat heart.

    PubMed

    Otani, H; Prasad, M R; Engelman, R M; Otani, H; Cordis, G A; Das, D K

    1988-11-01

    In this study, we examined phosphoinositide metabolism during ischemia and reperfusion using an isolated and perfused rat heart. When myocardial phosphoinositides were prelabeled with [3H]inositol, reperfusion after 30 minutes of normothermic global ischemia resulted in significant accumulations of radiolabeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly in the heart reperfused with [3H]inositol after 30 minutes of ischemia compared with that perfused with [3H]inositol after 30 minutes of nonischemic perfusion. However, isotopic incorporation of [3H]glycerol into diacylglycerol, phosphatidic acid, and all of the three phosphoinositides was diminished in the reperfused hearts. Reperfusion of the ischemic heart prelabeled with [14C]arachidonic acid resulted in significant increases in [14C]diacylglycerol and [14C]phosphatidic acid. The enhanced accumulations of [3H]inositol phosphates during reperfusion were not affected by treatment with prazosin plus atropine or indomethacin, but were inhibited by hypoxic reperfusion, reperfusion with Ca2+-free buffer, or by mepacrine. These results suggest that myocardial reperfusion stimulates phosphodiesteratic breakdown and turnover of phosphoinositides, and increased Ca2+ influx caused by reperfusion may be involved in the mechanism of stimulation of phosphatidylinositol-specific phospholipase C activity in the rat heart.

  18. Simultaneous inhibition of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways augment the sensitivity to actinomycin D in Ewing sarcoma.

    PubMed

    Yamamoto, Takatoshi; Ohno, Takatoshi; Wakahara, Kazuhiko; Nagano, Akihito; Kawai, Gou; Saitou, Mitsuru; Takigami, Iori; Matsuhashi, Aya; Yamada, Kazunari; Shimizu, Katsuji

    2009-08-01

    Ewing sarcoma cells, of which over 85% retain chimeric fusion gene EWS/Fli-1, are by and large more resistant to chemotherapeutics compared to nonneoplastic cells. The purpose of this study is to determine the role of EWS/Fli-1 fusion and its downstream targets regarding the cells' resistance against actinomycin D (ActD), which is one of the most commonly used antitumor agents in combination chemotherapy of Ewing sarcomas. Cytotoxicity was measured by WST-8 assay. Caspase-dependent and -independent cell death was examined by fluorescence microscope. Protein expression was analyzed by western blotting. Caspase activity was determined by Caspase-Glo assay. ActD-induced caspase-dependent apoptotic cell death to Ewing sarcoma TC-135 cells in a dose- and time- dependent manner. Knockdown of EWS/Fli-1 fusion by siRNA resulted in enhancement of ActD-induced apoptosis. ActD treatment activated both mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways although in a distinctive manner. Combined administration of U0126 (MEK inhibitor) and LY294002 (PI3K inhibitor) significantly enhanced ActD-induced apoptosis in vitro and suppressed xenograft tumor growth in vivo. The present study demonstrated for the first time that combination of U0126 and LY294002 can augment the cytotoxicity of ActD against Ewing sarcoma cells in vitro and in vivo. Our results indicate that further study on combination of conventional chemotherapies with MEK and PI3K inhibitors may be considered for innovative treatments of Ewing sarcoma patients.

  19. The Neuron-Specific Rai (ShcC) Adaptor Protein Inhibits Apoptosis by Coupling Ret to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Pelicci, Giuliana; Troglio, Flavia; Bodini, Alessandra; Melillo, Rosa Marina; Pettirossi, Valentina; Coda, Laura; De Giuseppe, Antonio; Santoro, Massimo; Pelicci, Pier Giuseppe

    2002-01-01

    Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals. PMID:12242309

  20. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

    PubMed Central

    Clarke, J F; Young, P W; Yonezawa, K; Kasuga, M; Holman, G D

    1994-01-01

    Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities. PMID:8010944

  1. Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

    PubMed

    Borradaile, Nica M; de Dreu, Linda E; Huff, Murray W

    2003-10-01

    The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr(-/-) mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo.

  2. Light-stimulated inositolphospholipid turnover in Samanea saman leaf pulvini

    SciTech Connect

    Morse, M.J.; Crain, R.C.; Satter, R.L.

    1987-10-01

    Leaflets of Samanea saman open and close rhythmically, driven by an endogenous circadian clock. Light has a rapid, direct effect on the movements and also rephases the rhythm. The authors investigated whether light signals might be mediated by increased inositolphospholipid turnover, a mechanism for signal transduction that is widely utilized in animal systems. Samanea motor organs (pulvini) labeled with (/sup 3/H)inositol were irradiated briefly (5-30 sec) with white light, and membrane-localized phosphatidylinositol phosphates and their aqueous breakdown products, the inositol phosphates, were examined. After a 15-sec or longer light pulse, labeled phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate decreased and their labeled metabolic products inositol 1,4-biphosphate and inositol 1,4,5-trisphosphate increased changes characteristic of inositolphospholipid turnover. The authors conclude that inositolphospholipid turnover may act as a phototransduction mechanism in Samanea pulvini in a manner that is similar to that reported in animal systems.

  3. Inhibition of Insulin Signaling in Endothelial Cells by Protein Kinase C-induced Phosphorylation of p85 Subunit of Phosphatidylinositol 3-Kinase (PI3K)*

    PubMed Central

    Maeno, Yasuhiro; Li, Qian; Park, Kyoungmin; Rask-Madsen, Christian; Gao, Benbo; Matsumoto, Motonobu; Liu, Yingjie; Wu, I-Hsien; White, Morris F.; Feener, Edward P.; King, George L.

    2012-01-01

    The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders. PMID:22158866

  4. Inhibition of p85, the non-catalytic subunit of phosphatidylinositol 3-kinase, exerts potent antitumor activity in human breast cancer cells

    PubMed Central

    Folgiero, V; Di Carlo, S E; Bon, G; Spugnini, E P; Di Benedetto, A; Germoni, S; Pia Gentileschi, M; Accardo, A; Milella, M; Morelli, G; Bossi, G; Mottolese, M; Falcioni, R

    2012-01-01

    The phosphoinositide 3-kinases (PI3Ks) are heterodimers consisting of the catalytic subunit p110 and the regulatory subunit p85. The PI3K/Akt pathway is strongly deregulated in breast cancer (BC) representing one of the mechanisms of resistance to therapies. Therefore, the identification of inhibitors of PI3K components represents one of the main goals to produce therapeutic agents. Here, we evaluated the efficacy of a phosphopeptide 1257 (P-1257) that targeting p85 strongly inhibits PI3K activity. We tested the effects of P-1257 administration in vitro and in vivo using BC cells expressing different levels of ErbB-2 and resistant or responsive to Trastuzumab. We demonstrated that inhibition of p85 activity by P-1257 induces cell death and sensitizes JIMT-1 and KPL-4 ErbB-2-overexpressing BC cells to Trastuzumab treatment. It is noteworthy that P-1257 delivery in vivo by electroporation or liposomes significantly inhibits the proliferation of tumor cells engrafted at subcutaneous and visceral sites. Overall, our data indicate that the p85 subunit is a valid target for therapeutic approaches and suggest that the structure of the peptide used in our study could be utilized for the development of novel drugs to apply in combination with therapies that fail to cure BCs with high PI3K activity. PMID:23222510

  5. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    PubMed

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis

  6. Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

    PubMed Central

    Ballou, L R; Barker, S C; Postlethwaite, A E; Kang, A H

    1991-01-01

    IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol. PMID:1845871

  7. The thiamine biosynthetic enzyme ThiC catalyzes multiple turnovers and is inhibited by S-adenosylmethionine (AdoMet) metabolites.

    PubMed

    Palmer, Lauren D; Downs, Diana M

    2013-10-18

    ThiC (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase; EC 4.1.99.17) is a radical S-adenosylmethionine (AdoMet) enzyme that uses a [4Fe-4S](+) cluster to reductively cleave AdoMet to methionine and a 5'-deoxyadenosyl radical that initiates catalysis. In plants and bacteria, ThiC converts the purine intermediate 5-aminoimidazole ribotide to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, an intermediate of thiamine pyrophosphate (coenzyme B1) biosynthesis. In this study, assay conditions were implemented that consistently generated 5-fold molar excess of HMP, demonstrating that ThiC undergoes multiple turnovers. ThiC activity was improved by in situ removal of product 5'-deoxyadenosine. The activity was inhibited by AdoMet metabolites S-adenosylhomocysteine, adenosine, 5'-deoxyadenosine, S-methyl-5'-thioadenosine, methionine, and homocysteine. Neither adenosine nor S-methyl-5'-thioadenosine had been shown to inhibit radical AdoMet enzymes, suggesting that ThiC is distinct from other family members. The parameters for improved ThiC activity and turnover described here will facilitate kinetic and mechanistic analyses of ThiC.

  8. Cancer-testis antigen MAGE-C2 binds Rbx1 and inhibits ubiquitin ligase-mediated turnover of cyclin E.

    PubMed

    Hao, Jiaqing; Song, Xiao; Wang, Jingjing; Guo, Chengli; Li, Yan; Li, Bing; Zhang, Yu; Yin, Yanhui

    2015-12-08

    Cancer-testis antigen MAGE-C2 is normally expressed in testis but aberrantly expressed in various kinds of tumors. Its functions in tumor cells are mostly unknown. Here, we show that MAGE-C2 binds directly to the RING domain protein Rbx1, and participates in Skp1-Cullin1-F box protein (SCF) complex. Furthermore, MAGE-C2 can inhibit the E3 ubiquitin ligase activity of SCF complex. Ablation of endogenous MAGE-C2 decreases the level of cyclin E and accelerates cyclin E turnover by inhibiting ubiquitin-mediated proteasome degradation. Overexpression of MAGE-C2 increases the level of cyclin E and promotes G1-S transition and cell proliferation, and the results are further confirmed by knockdown of MAGE-C2. Overall, the study indicates that MAGE-C2 is involved in SCF complex and increases the stability of cyclin E in tumor cells.

  9. Cancer-testis antigen MAGE-C2 binds Rbx1 and inhibits ubiquitin ligase-mediated turnover of cyclin E

    PubMed Central

    Wang, Jingjing; Guo, Chengli; Li, Yan; Li, Bing; Zhang, Yu; Yin, Yanhui

    2015-01-01

    Cancer-testis antigen MAGE-C2 is normally expressed in testis but aberrantly expressed in various kinds of tumors. Its functions in tumor cells are mostly unknown. Here, we show that MAGE-C2 binds directly to the RING domain protein Rbx1, and participates in Skp1-Cullin1-F box protein (SCF) complex. Furthermore, MAGE-C2 can inhibit the E3 ubiquitin ligase activity of SCF complex. Ablation of endogenous MAGE-C2 decreases the level of cyclin E and accelerates cyclin E turnover by inhibiting ubiquitin-mediated proteasome degradation. Overexpression of MAGE-C2 increases the level of cyclin E and promotes G1-S transition and cell proliferation, and the results are further confirmed by knockdown of MAGE-C2. Overall, the study indicates that MAGE-C2 is involved in SCF complex and increases the stability of cyclin E in tumor cells. PMID:26540345

  10. Turnover Time

    EPA Science Inventory

    Ecosystems contain energy and materials such as carbon, nitrogen, phosphorus, and water, and are open to their flow-through. Turnover time refers to the amount of time required for replacement by flow-through of the energy or substance of interest contained in the system, and is ...

  11. Turnover Time

    EPA Science Inventory

    Ecosystems contain energy and materials such as carbon, nitrogen, phosphorus, and water, and are open to their flow-through. Turnover time refers to the amount of time required for replacement by flow-through of the energy or substance of interest contained in the system, and is ...

  12. Phosphatidylinositol hydrolysis in isolated guinea-pig islets of Langerhans.

    PubMed Central

    Schrey, M P; Montague, W

    1983-01-01

    Previous studies have reported an increased turnover of phospholipid in isolated islets of Langerhans in response to raised glucose concentrations. The present investigation was thus undertaken to determine the nature of any phospholipases that may be implicated in this phenomenon by employing various radiolabelled exogenous phospholipids. Hydrolysis of 1-acyl-2-[14C]arachidonoylglycerophosphoinositol by a sonicated preparation of islets optimally released radiolabelled lysophosphatidylinositol, arachidonic acid and 1,2-diacylglycerol at pH 5,7 and 9 respectively. This indicates the presence of a phospholipase A1 and a phospholipase C. However, the lack of any labelled lysophosphatidylinositol production when 2-acyl-1-[14C]stearoylglycerophosphoinositol was hydrolysed argues against a role for phospholipase A2 in the release of arachidonic acid. Phospholipase C activity as measured by phosphatidyl-myo-[3H]inositol hydrolysis was optimal around pH8, required Ca2+ for activity and was predominantly cytosolic in origin. The time course of phosphatidylinositol hydrolysis at pH 6 indicated a precursor-product relationship for 1,2-diacylglycerol and arachidonic acid respectively. The release of these two products when phosphatidylinositol was hydrolysed by either islet or acinar tissue was similar. However, phospholipase A1 activity was 20-fold higher in acinar tissue. Substrate specificity studies with islet tissue revealed that arachidonic acid release from phosphatidylethanolamine and phosphatidylcholine was only 8% and 2.5% respectively of that from phosphatidylinositol. Diacylglycerol lipase was also demonstrated in islet tissue being predominantly membrane bound and stimulated by Ca2+. The availability of non-esterified arachidonic acid in islet cells could be regulated by changes in the activity of a phosphatidylinositol-specific phospholipase C acting in concert with a diacylglycerol lipase. PMID:6362663

  13. Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor-Mediated Signaling in Neuroblastoma.

    PubMed

    Soori, Mehrnoosh; Lu, Guizhen; Mason, Robert W

    2016-02-01

    Inhibition of the major lysosomal proteases, cathepsins B, D, and L, impairs growth of several cell types but leads to apoptosis in neuroblastoma. The goal of this study was to examine the mechanisms by which enzyme inhibition could cause cell death. Cathepsin inhibition caused cellular accumulation of fragments of the insulin growth factor 1 (IGF-1) receptor. The fragments were located in dense organelles that were characterized as autophagosomes. This novel discovery provides the first clear link between lysosomal function, autophagy, and IGF-1- mediated cell proliferation. A more in-depth analysis of the IGF1 signaling pathway revealed that the mitogen-activated protein kinase (MAPK) cell-proliferation pathway was impaired in inhibitor treated cells, whereas the Akt cell survival pathway remained functional. Shc, an adapter protein that transmits IGF-1 signaling through the MAPK pathway, was sequestered in autophagosomes; whereas IRS-2, an adapter protein that transmits IGF-1 signaling through the Akt pathway, was unaffected by cathepsin inhibition. Furthermore, Shc was sequestered in autophagosomes as its active form, indicating that autophagy is a key mechanism for downregulating IGF-1-induced cell proliferation. Cathepsin inhibition had a greater effect on autophagic sequestration of the neuronal specific adapter protein, Shc-C, than ubiquitously expressed Shc-A, providing mechanistic support for the enhanced sensitivity of neuronally derived tumor cells. We also observed impaired activation of MAPK by epidermal growth factor treatment in inhibitor-treated cells. The Shc adapter proteins are central to transducing proliferation signaling by a range of receptor tyrosine kinases; consequently, cathepsin inhibition may become an important therapeutic approach for treating neuroblastoma and other tumors of neuronal origin.

  14. Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor–Mediated Signaling in Neuroblastoma

    PubMed Central

    Soori, Mehrnoosh; Lu, Guizhen

    2016-01-01

    Inhibition of the major lysosomal proteases, cathepsins B, D, and L, impairs growth of several cell types but leads to apoptosis in neuroblastoma. The goal of this study was to examine the mechanisms by which enzyme inhibition could cause cell death. Cathepsin inhibition caused cellular accumulation of fragments of the insulin growth factor 1 (IGF-1) receptor. The fragments were located in dense organelles that were characterized as autophagosomes. This novel discovery provides the first clear link between lysosomal function, autophagy, and IGF-1– mediated cell proliferation. A more in-depth analysis of the IGF1 signaling pathway revealed that the mitogen-activated protein kinase (MAPK) cell-proliferation pathway was impaired in inhibitor treated cells, whereas the Akt cell survival pathway remained functional. Shc, an adapter protein that transmits IGF-1 signaling through the MAPK pathway, was sequestered in autophagosomes; whereas IRS-2, an adapter protein that transmits IGF-1 signaling through the Akt pathway, was unaffected by cathepsin inhibition. Furthermore, Shc was sequestered in autophagosomes as its active form, indicating that autophagy is a key mechanism for downregulating IGF-1-induced cell proliferation. Cathepsin inhibition had a greater effect on autophagic sequestration of the neuronal specific adapter protein, Shc-C, than ubiquitously expressed Shc-A, providing mechanistic support for the enhanced sensitivity of neuronally derived tumor cells. We also observed impaired activation of MAPK by epidermal growth factor treatment in inhibitor-treated cells. The Shc adapter proteins are central to transducing proliferation signaling by a range of receptor tyrosine kinases; consequently, cathepsin inhibition may become an important therapeutic approach for treating neuroblastoma and other tumors of neuronal origin. PMID:26660229

  15. Changes in expression of cell wall turnover genes accompany inhibition of chromosome segregation by bovine protein kinase C alpha expression in Saccharomyces cerevisiae.

    PubMed

    Sprowl, Jason A; Villeneuve, David J; Guo, Baoqing; Young, Andrew J M; Hembruff, Stacey L; Parissenti, Amadeo M

    2007-10-01

    Expression of bovine PKCalpha in Saccharomyces cerevisiae results in growth inhibition, which is strongly augmented upon addition of phorbol esters. To investigate the nature of this PKC-induced inhibition of cell growth, wildtype and bovine PKCalpha-expressing yeast cells were examined by flow cytometry and by fluorescence microscopy after staining with propidium iodide. Upon expression and activation of the mammalian PKC isoform, cells accumulated in the G2/M phase of the cell cycle and exhibited impaired chromsome segregation. In some instances, PKC expression and activation was accompanied by a defect in septum formation between mother and daughter cells. cDNA microarray analysis revealed 4 genes (CTS1, DSE1, DSE2, and SVS1) that changed expression in both a PKCalpha- and phorbol ester-dependent manner. These findings were confirmed by quantitative real-time PCR. Three of these genes are involved in cell wall turnover and are regulated by a single transcription factor (Ace 2) that localizes to daughter cell nuclei after cytokinesis. Taken together, these observations suggest that expression and activation of bovine PKCalpha in yeast cells repress growth by inducing an accumulation of cells in G2/M, likely through an impairment of chromosome segregation, cytokinesis, and septum formation. Moreover, when these observations are taken in the context of previously published observations with various yeast null mutants, we propose that bovine PKCalpha may directly or indirectly activate a subunit of the PP2A phosphatase complex (cdc55), which is a component of the mitotic spindle checkpoint.

  16. Pseudopterosin A inhibits phagocytosis and alters intracellular calcium turnover in a pertussis toxin sensitive site in Tetrahymena thermophila.

    PubMed

    Moya, Claudia E; Jacobs, Robert S

    2006-08-01

    The free living ciliate Tetrahymena thermophila was chosen as a cellular model in order to investigate the mode of action of the anti-inflammatory marine natural product Pseudopterosin A (PsA). In this paper we present evidence that PsA inhibits phagosome formation (KD=10.5 microM) and triggers a discrete intracellular calcium release (depletion) from a site in T. thermophila cells (KD=6.4 microM). Pre-treatment with the Gi/o protein inhibitor, pertussis toxin (PTX), inhibits PsA activity of both responses providing pharmacological evidence that the site of action for PsA is at a PTX sensitive G protein or a G protein coupled receptor (GPCR). Addition of extracellular calcium induced a concentration dependent increase in the incidence of phagosome formation (KD=30.3 microM) and was blocked by PsA pre-treatment. This particular effect of PsA on extracellular calcium was not blocked by PTX pre-treatment.

  17. Phosphatidic acid stimulates cardiac KATP channels like phosphatidylinositols, but with novel gating kinetics.

    PubMed

    Fan, Zheng; Gao, Lizhi; Wang, Wenxia

    2003-01-01

    Membrane-bound anionic phospholipids such as phosphatidylinositols have the capacity to modulate ATP-sensitive potassium (K(ATP)) channels through a mechanism involving long-range electrostatic interaction between the lipid headgroup and channel. However, it has not yet been determined whether the multiple effects of phosphatidylinositols reported in the literature all result from this general electrostatic interaction or require a specific headgroup structure. The present study investigated whether phosphatidic acid (PA), an anionic phospholipid substantially different in structure from phosphatidylinositols, evokes effects similar to phosphatidylinositols on native K(ATP) channels of rat heart and heterogeneous Kir6.2/SUR2A channels. Channels treated with PA (0.2-1 mg/ml applied to the cytoplasmic side of the membrane) exhibited higher activity, lower sensitivity to ATP inhibition, less Mg(2+)-dependent nucleotide stimulation, and poor sulfonylurea inhibition. These effects match the spectrum of phosphatidylinositols' effects, but, in addition, PA also induced a novel pattern in gating kinetics, represented by a decreased mean open time (from 12.2 +/- 2.0 to 3.3 +/- 0.7 ms). This impact on gating kinetics clearly distinguishes PA's effects from those of phosphatidylinositols. Results indicate that multiple effects of anionic phospholipids on K(ATP) channels are related phenomena and can likely be attributed to a common mechanism, but additional specific effects due to other mechanisms may also coincide.

  18. Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M-current inhibition

    PubMed Central

    del Río, Elena; Bevilacqua, Jorge A; Marsh, Stephen J; Halley, Pamela; Caulfield, Malcolm P

    1999-01-01

    The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M-current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. Phosphoinositide-specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]-cytidine monophosphate phosphatidate (CMP-PA) after incubation with [3H]-cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo-M) stimulated PLC in a dose-dependent manner with an EC50 of approximately 3.5 μm. The concentration-response curve for oxo-M was shifted to the right by a factor of about 10 by pirenzepine (100 nm), suggesting a pKB (—log of the apparent dissociation constant) of 7.9 ± 0.4, while himbacine (1 μm) shifted the curve by a factor of about 13 (pKB∼7.1 ± 0.6). This indicates involvement of the M1 muscarinic receptor in this response. The accumulation of CMP-PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. The ability of oxo-M to release Ca2+ from inositol(1,4,5)trisphosphate (InsP3)-sensitive stores was determined by fura-2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo-M failed to evoke intracellular Ca2+ (Cai2+) mobilisation in SCG neurones voltage clamped at −60 mV, but produced a significant Cai2+ rise (67 ± 15 nm, n = 9) in cells voltage clamped at −25 mV. Thapsigargin (0.5–1 μm) caused a 70% inhibition of the oxo-M-induced Cai2+ increase, indicating its intracellular origin, while oxo-M-induced inhibition of M-current in the same cells was unaffected by thapsigargin. Our results do not support the involvement of InsP3-sensitive calcium mobilisation in M-current inhibition. PMID:10517804

  19. E6AP inhibits G-CSFR turnover and functions by promoting its ubiquitin-dependent proteasome degradation.

    PubMed

    Chhabra, Stuti; Kumar, Yogesh; Thacker, Gatha; Kapoor, Isha; Lochab, Savita; Sanyal, Sabyasachi; Bhatt, Madan L B; Chattopadhyay, Naibedya; Trivedi, Arun Kumar

    2017-10-01

    Granulocyte colony-stimulating factor receptor (G-CSFR) plays a crucial role in regulating myeloid cell survival, proliferation, and neutrophilic granulocyte precursor cells maturation. Previously, we demonstrated that Fbw7α negatively regulates G-CSFR and its downstream signaling through ubiquitin-proteasome mediated degradation. However, whether additional ubiquitin ligases for G-CSFR exist is not known. Identifying multiple E3 ubiquitin ligases for G-CSFR shall improve our understanding of activation and subsequent attenuation of G-CSFR signaling required for differentiation and proliferation. Here, for the first time we demonstrate that E6 associated protein (E6AP), an E3 ubiquitin ligase physically associates with G-CSFR and targets it for ubiquitin-mediated proteasome degradation and thereby attenuates its functions. We further show that E6AP promoted G-CSFR degradation leads to reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3) which is required for G-CSF dependent granulocytic differentiation. More importantly, our finding shows that E6AP also targets mutant form of G-SCFR (G-CSFR-T718), frequently observed in severe congenital neutropenia (SCN) patients that very often culminate to AML, however, at a quite slower rate than wild type G-CSFR. In addition, our data showed that knockdown of E6AP restores G-CSFR and its signaling thereby promoting granulocytic differentiation. Collectively, our data demonstrates that E6AP facilitates ubiquitination and subsequent degradation of G-CSFR leading to attenuation of its downstream signaling and inhibition of granulocytic differentiation. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. A turnover study of hypothalamic monoamine oxidase (MAO) and effects of MAO inhibition on gonadotropin secretion in the female catfish, Heteropneustes fossilis.

    PubMed

    Senthilkumaran, B; Joy, K P

    1995-01-01

    In the present study, the rate constants of degradation (k) and synthesis (S) and half-life of hypothalamic monoamine oxidase were determined to explain annual variations and biphasic effects of the enzyme to low and high doses of estradiol-17 beta (E2) in 3-week ovariectomized Heteropneustes fossilis. In the preparatory phase, the half-life (t1/2) of the enzyme was the longest (21.16 days) with low k (0.03275 days-1) and S (0.000845 Units/day) values, suggesting a low turnover of the enzyme. In the prespawning phase the t1/2 was the shortest (11.65 days) with high k (0.0595 days-1) and S (0.011 Units/day) values. The low and high turnovers of the enzyme, respectively, in these two seasons could be correlated to low and high profiles of plasma E2 levels. In the resting phase, the values were in between (t1/2 = 18.83 days, k = 0.0368 days-1, S = 0.00211 Units/day) but the plasma E2 level was undetectable. Three weeks of ovariectomy increased the t1/2 (19.04 days) compared to that of the control (11.61 days) with decreases in both k and S values. The administration of a low dose of E2 (0.1 micrograms/g BW) further increased the t1/2 (19.63 days) over that of the ovariectomized fish with a significant rise in the S value. However, a high dose of E2 (1.0 micrograms/g BW) decreased it (13.33 days) by reducing the S and elevating the k values. These results suggest that the stimulatory effect of low doses of E2 on the enzyme activity is produced by elevating its synthesis rate and the inhibitory effect of high doses of E2 by simultaneously decreasing the synthesis and increasing the degradation rates of the enzyme. The administration of a single dose (75 mg/kg BW) of pargyline has elevated plasma gonadotropin (GTH) level after 3 and 6 hr and 7 days in the sham-ovariectomized (control), ovariectomized, and ovariectomized low-E2-dose groups; the peak increase was found at 6 hr. On the contrary, in the ovariectomized high-E2 group the GTH level was inhibited at 3 and 6 hr

  1. ISA-2011B, a Phosphatidylinositol 4-Phosphate 5-Kinase α Inhibitor, Impairs CD28-Dependent Costimulatory and Pro-inflammatory Signals in Human T Lymphocytes

    PubMed Central

    Kunkl, Martina; Porciello, Nicla; Mastrogiovanni, Marta; Capuano, Cristina; Lucantoni, Federica; Moretti, Chiara; Persson, Jenny L.; Galandrini, Ricciarda; Buzzetti, Raffaella; Tuosto, Loretta

    2017-01-01

    Phosphatidylinositol 4,5-biphosphate (PIP2) is a membrane phospholipid that controls the activity of several proteins regulating cytoskeleton reorganization, cytokine gene expression, T cell survival, proliferation, and differentiation. Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are the main enzymes involved in PIP2 biosynthesis by phosphorylating phosphatidylinositol 4-monophosphate (PI4P) at the D5 position of the inositol ring. In human T lymphocytes, we recently found that CD28 costimulatory molecule is pivotal for PIP2 turnover by recruiting and activating PIP5Kα. We also found that PIP5Kα is the main regulator of both CD28 costimulatory signals integrating those delivered by TCR as well as CD28 autonomous signals regulating the expression of pro-inflammatory genes. Given emerging studies linking alterations of PIP2 metabolism to immune-based diseases, PIP5Kα may represent a promising target to modulate immunity and inflammation. Herewith, we characterized a recently discovered inhibitor of PIP5Kα, ISA-2011B, for its inhibitory effects on T lymphocyte functions. We found that the inhibition of PIP5Kα lipid-kinase activity by ISA-2011B significantly impaired CD28 costimulatory signals necessary for TCR-mediated Ca2+ influx, NF-AT transcriptional activity, and IL-2 gene expression as well as CD28 autonomous signals regulating the activation of NF-κB and the transcription of pro-inflammatory cytokine and chemokine genes. Moreover, our data on the inhibitory effects of ISA-2011B on CD28-mediated upregulation of inflammatory cytokines related to Th17 cell phenotype in type 1 diabetes patients suggest ISA-2011B as a promising anti-inflammatory drug. PMID:28491063

  2. Stimulation of inorganic-phosphate incorporation into phosphatidylinositol in rat thoracic aorta mediated through V1-vasopressin receptors.

    PubMed Central

    Takhar, A P; Kirk, C J

    1981-01-01

    Vasopressin stimulates the incorporation of [32P]Pi into phosphatidylinositol but not into other phospholipids in rat thoracic aorta strips. The relative abilities of three vasopressin analogues to stimulate phosphatidylinositol labelling in rat aorta are similar to their relative pressor potencies in vivo and to their relative potencies in stimulating the metabolism of rat hepatocytes, but very different from their relative antidiuretic potencies. The vasopressor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin competitively inhibits [Arg8]vasopressin-stimulated phosphatidylinositol labelling in rat aorta with a pA2 of 8.1. It is concluded that the Ca2+-mobilizing vasopressin receptors (V1-receptors) of the rat aorta stimulate phosphatidylinositol metabolism, probably by enhancing phosphatidylinositol breakdown. PMID:6272723

  3. Bimodal lipid substrate dependence of phosphatidylinositol kinase.

    PubMed

    Ganong, B R

    1990-07-24

    Phosphatidylinositol (PI) kinase activity was solubilized from rat liver microsomes and partially purified by chromatography on hydroxyapatite and Reactive Green 19-Superose. Examination of the ATP dependence using a mixed micellar assay gave a Km of 120 microM. The dependence of reaction rate on PI was more complicated. PI kinase bound a large amount of Triton X-100, and as expected for a micelle-associated enzyme utilizing a micelle-associated lipid substrate, the reaction rate was dependent on the micellar mole fraction, PI/(PI + Triton X-100), with a Km of 0.02 (unitless). Activity showed an additional dependence on bulk PI concentration at high micelle dilution. These results demonstrated two kinetically distinguishable steps leading to formation of a productive PI/enzyme(/ATP) complex. The rate of the first step, which probably represents exchange of PI from the bulk micellar pool into enzyme-containing micelles, depends on bulk PI concentration. The rate of the second step, association of PI with enzyme within a single micelle, depends on the micellar mole fraction of PI. Depression of the apparent Vmax at low ionic strength suggested that electrostatic repulsion between negatively charged PI/Triton X-100 mixed micelles inhibits PI exchange, consistent with a model in which intermicellar PI exchange depends on micellar collisions.

  4. 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.

  5. 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

  6. Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils

    SciTech Connect

    Traynor-Kaplan, A.E.; Thompson, B.L.; Harris, A.L.; Taylor, P.; Omann, G.M.; Sklar, L.A. )

    1989-09-15

    We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.

  7. Ugi Reaction-Derived α-Acyl Aminocarboxamides Bind to Phosphatidylinositol 3-Kinase-Related Kinases, Inhibit HSF1-Dependent Heat Shock Response, and Induce Apoptosis in Multiple Myeloma Cells.

    PubMed

    Bach, Matthias; Lehmann, Anna; Brünnert, Daniela; Vanselow, Jens T; Hartung, Andreas; Bargou, Ralf C; Holzgrabe, Ulrike; Schlosser, Andreas; Chatterjee, Manik

    2017-05-25

    Heat shock transcription factor 1 (HSF1) has been identified as a therapeutic target for pharmacological treatment of multiple myeloma (MM). However, direct therapeutic targeting of HSF1 function seems to be difficult due to the shortage of clinically suitable pharmacological inhibitors. We utilized the Ugi multicomponent reaction to create a small but smart library of α-acyl aminocarboxamides and evaluated their ability to suppress heat shock response (HSR) in MM cells. Using the INA-6 cell line as the MM model and the strictly HSF1-dependent HSP72 induction as a HSR model, we identified potential HSF1 inhibitors. Mass spectrometry-based affinity capture experiments with biotin-linked derivatives revealed a number of target proteins and complexes, which exhibit an armadillo domain. Also, four members of the tumor-promoting and HSF1-associated phosphatidylinositol 3-kinase-related kinase (PIKK) family were identified. The antitumor activity was evaluated, showing that treatment with the anti-HSF1 compounds strongly induced apoptotic cell death in MM cells.

  8. Targeting Plasmodium phosphatidylinositol 4-kinase to eliminate malaria

    PubMed Central

    Lim, Chek Shik; Lim, Siau Hoi; Roland, Jason; Simon, Oliver; Yeung, Bryan KS; Chatterjee, Arnab K; McCormack, Susan L; Manary, Micah J; Zeeman, Anne-Marie; Dechering, Koen J; Kumar, TR Santha; Henrich, Philipp P; Gagaring, Kerstin; Ibanez, Maureen; Kato, Nobutaka; Kuhen, Kelli L; Fischli, Christoph; Nagle, Advait; Rottmann, Matthias; Plouffe, David M; Bursulaya, Badry; Meister, Stephan; Rameh, Lucia; Trappe, Joerg; Haasen, Dorothea; Timmerman, Martijn; Sauerwein, Robert W; Suwanarusk, Rossarin; Russell, Bruce; Renia, Laurent; Nosten, Francois; Tully, David C; Kocken, Clemens HM; Glynne, Richard J; Bodenreider, Christophe; Fidock, David A; Diagana, Thierry T; Winzeler, Elizabeth A

    2014-01-01

    Summary Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here, we identify a lipid kinase, phosphatidylinositol 4-kinase (PI4K), as the target of imidazopyrazines, a novel antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens, P. falciparum and P. vivax, and inhibit liver stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI4K, altering the intracellular distribution of phosphatidylinositol 4-phosphate. Collectively, our data define PI4K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria. PMID:24284631

  9. Biochemical and genetic evidence for the presence of multiple phosphatidylinositol- and phosphatidylinositol 4,5-bisphosphate-specific phospholipases C in Tetrahymena.

    PubMed

    Leondaritis, George; Sarri, Theoni; Dafnis, Ioannis; Efstathiou, Antonia; Galanopoulou, Dia

    2011-03-01

    Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], produce the Ca(2+)-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca(2+). One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca(2+), modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P(2) levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P(3)-Ca(2+) regulatory axis in ciliates.

  10. Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena▿‡

    PubMed Central

    Leondaritis, George; Sarri, Theoni; Dafnis, Ioannis; Efstathiou, Antonia; Galanopoulou, Dia

    2011-01-01

    Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], produce the Ca2+-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca2+. One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca2+, modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P2 levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P3-Ca2+ regulatory axis in ciliates. PMID:21169416

  11. Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in Arabidopsis.

    PubMed

    Okazaki, Kumiko; Miyagishima, Shin-ya; Wada, Hajime

    2015-03-01

    Chloroplast division is performed by the constriction of envelope membranes at the division site. Although constriction of a ring-like protein complex has been shown to be involved in chloroplast division, it remains unknown how membrane lipids participate in the process. Here, we show that phosphoinositides with unknown function in envelope membranes are involved in the regulation of chloroplast division in Arabidopsis thaliana. PLASTID DIVISION1 (PDV1) and PDV2 proteins interacted specifically with phosphatidylinositol 4-phosphate (PI4P). Inhibition of phosphatidylinositol 4-kinase (PI4K) decreased the level of PI4P in chloroplasts and accelerated chloroplast division. Knockout of PI4Kβ2 expression or downregulation of PI4Kα1 expression resulted in decreased levels of PI4P in chloroplasts and increased chloroplast numbers. PI4Kα1 is the main contributor to PI4P synthesis in chloroplasts, and the effect of PI4K inhibition was largely abolished in the pdv1 mutant. Overexpression of DYNAMIN-RELATED PROTEIN5B (DRP5B), another component of the chloroplast division machinery, which is recruited to chloroplasts by PDV1 and PDV2, enhanced the effect of PI4K inhibition, whereas overexpression of PDV1 and PDV2 had additive effects. The amount of DRP5B that associated with chloroplasts increased upon PI4K inhibition. These findings suggest that PI4P is a regulator of chloroplast division in a PDV1- and DRP5B-dependent manner. © 2015 American Society of Plant Biologists. All rights reserved.

  12. The Yeast Bsd2-1 Mutation Influences Both the Requirement for Phosphatidylinositol Transfer Protein Function and Derepression of Phospholipid Biosynthetic Gene Expression in Yeast

    PubMed Central

    Kagiwada, S.; Kearns, B. G.; McGee, T. P.; Fang, M.; Hosaka, K.; Bankaitis, V. A.

    1996-01-01

    The BSD2-1 allele renders Saccharomyces cerevisiae independent of its normally essential requirement for phosphatidylinositol transfer protein (Sec14p) in the stimulation of Golgi secretory function and cell viability. We now report that BSD2-1 yeast mutants also exhibit yet another phenotype, an inositol auxotrophy. We demonstrate that the basis for this Ino(-) phenotype is the inability of BSD2-1 strains to derepress transcription of INO1, the structural gene for the enzyme that catalyzes the committed step in de novo inositol biosynthesis in yeast. This constitutive repression of INO1 expression is mediated through specific inactivation of Ino2p, a factor required for trans-activation of INO1 transcription, and we show that these transcriptional regulatory defects can be uncoupled from the ``bypass Sec14p'' phenotype of BSD2-1 strains. Finally, we present evidence that newly synthesized phosphatidylinositol is subject to accelerated turnover in BSD2-1 mutants and that prevention of this accelerated phosphatidylinositol turnover in turn negates suppression of Sec14p defects by BSD2-1. We propose that, in BSD2-1 strains, a product(s) generated by phosphatidylinositol turnover coordinately modulates the activities of both the Sec14p/Golgi pathway and the pathway through which transcription of phospholipid biosynthetic genes is derepressed. PMID:8725219

  13. CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8(+) T cells.

    PubMed

    Strachan, Debbie C; Ruffell, Brian; Oei, Yoko; Bissell, Mina J; Coussens, Lisa M; Pryer, Nancy; Daniel, Dylan

    2013-12-01

    Increased numbers of tumor-infiltrating macrophages correlate with poor disease outcome in patients affected by several types of cancer, including breast and prostate carcinomas. The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the neoplastic microenvironment and promotes the differentiation of TAMs toward a pro-tumorigenic phenotype. Twelve clinical trials are currently evaluating agents that target the CSF1/CSF1R signaling pathway as a treatment against multiple malignancies, including breast carcinoma, leukemia, and glioblastoma. The blockade of CSF1R signaling has been shown to greatly decrease the number of macrophages in a tissue-specific manner. However, additional mechanistic insights are needed in order to understand how macrophages are depleted and the global effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using BLZ945, a highly selective small molecule inhibitor of CSF1R, we show that CSF1R inhibition attenuates the turnover rate of TAMs while increasing the number of CD8(+) T cells that infiltrate cervical and breast carcinomas. Specifically, we find that BLZ945 decreased the growth of malignant cells in the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis. Furthermore, we show that BLZ945 prevents tumor progression in the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis. Our results demonstrate that TAMs undergo a constant turnover in a CSF1R-dependent manner, and suggest that continuous inhibition of the CSF1R pathway may be essential to maintain efficacious macrophage depletion as an anticancer therapy.

  14. CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8+ T cells

    PubMed Central

    Strachan, Debbie C; Ruffell, Brian; Oei, Yoko; Bissell, Mina J; Coussens, Lisa M; Pryer, Nancy; Daniel, Dylan

    2013-01-01

    Increased numbers of tumor-infiltrating macrophages correlate with poor disease outcome in patients affected by several types of cancer, including breast and prostate carcinomas. The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the neoplastic microenvironment and promotes the differentiation of TAMs toward a pro-tumorigenic phenotype. Twelve clinical trials are currently evaluating agents that target the CSF1/CSF1R signaling pathway as a treatment against multiple malignancies, including breast carcinoma, leukemia, and glioblastoma. The blockade of CSF1R signaling has been shown to greatly decrease the number of macrophages in a tissue-specific manner. However, additional mechanistic insights are needed in order to understand how macrophages are depleted and the global effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using BLZ945, a highly selective small molecule inhibitor of CSF1R, we show that CSF1R inhibition attenuates the turnover rate of TAMs while increasing the number of CD8+ T cells that infiltrate cervical and breast carcinomas. Specifically, we find that BLZ945 decreased the growth of malignant cells in the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis. Furthermore, we show that BLZ945 prevents tumor progression in the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis. Our results demonstrate that TAMs undergo a constant turnover in a CSF1R-dependent manner, and suggest that continuous inhibition of the CSF1R pathway may be essential to maintain efficacious macrophage depletion as an anticancer therapy. PMID:24498562

  15. The effects of barbiturates on the metabolism of phosphatidic acid and phosphatidylinositol in rat brain synaptosomes.

    PubMed Central

    Miller, J C; Leung, I

    1979-01-01

    Barbiturates and diphenylhydantoin inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol and phosphatidic acid, but have a relatively slight effect on the incorporation of 32P into these lipids in the absence of carbamoylcholine and no effect on 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. Inhibition of the carbamoylcholine-stimulated increase was observed for pentobarbital, thiopental, phenobarbital, 5-(1,3-dimethylbutyl)-5-ethylbarbiturate, (+)- and (-)-5-ethyl-N-methyl-5-propylbarbituate and diphenylhydantoin. Similar concentrations of barbiturates and diphenylhydantoin were previously reported to inhibit the K+-stimulated Ca2+ influx, and therefore other agents that affect Ca2+ influx were tested to find whether they had any effect on 32P incorporation into these lipids. K+ (35 mM) increases 32P incorporation into phosphatidic acid, but to a smaller degree than 100 micrometer-carbamoylcholine, and its effect was inhibited by pentobarbital. Veratridine (75 micrometer) does not increase 32P incorporation into either phosphatidic acid or phosphatidylinositol, but did inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol. The possible relationship between the phospholipid effect and stimulated Ca2+ influx is discussed. PMID:435289

  16. Altered bone turnover during spaceflight

    NASA Technical Reports Server (NTRS)

    Turner, R. T.; Morey, E. R.; Liu, C.; Baylink, D. J.

    1982-01-01

    Modifications in calcium metabolism during spaceflight were studied, using parameters that reflect bone turnover. Bone formation rate, medullary area, bone length, bone density, pore size distribution, and differential bone cell number were evaluated in growing rate both immediately after and 25 days after orbital spaceflights aboard the Soviet biological satellites Cosmos 782 and 936. The primary effect of space flight on bone turnover was a reversible inhibition of bone formation at the periosteal surface. A simultaneous increase in the length of the periosteal arrest line suggests that bone formation ceased along corresponding portions of that surface. Possible reasons include increased secretion of glucocorticoids and mechanical unloading of the skeleton due to near-weightlessness, while starvation and immobilization are excluded as causes.

  17. Altered bone turnover during spaceflight

    NASA Technical Reports Server (NTRS)

    Turner, R. T.; Morey, E. R.; Liu, C.; Baylink, D. J.

    1982-01-01

    Modifications in calcium metabolism during spaceflight were studied, using parameters that reflect bone turnover. Bone formation rate, medullary area, bone length, bone density, pore size distribution, and differential bone cell number were evaluated in growing rate both immediately after and 25 days after orbital spaceflights aboard the Soviet biological satellites Cosmos 782 and 936. The primary effect of space flight on bone turnover was a reversible inhibition of bone formation at the periosteal surface. A simultaneous increase in the length of the periosteal arrest line suggests that bone formation ceased along corresponding portions of that surface. Possible reasons include increased secretion of glucocorticoids and mechanical unloading of the skeleton due to near-weightlessness, while starvation and immobilization are excluded as causes.

  18. Combined Inhibition of Both p110α and p110β Isoforms of Phosphatidylinositol 3-Kinase Is Required for Sustained Therapeutic Effect in PTEN-Deficient, ER(+) Breast Cancer.

    PubMed

    Hosford, Sarah R; Dillon, Lloye M; Bouley, Stephanie J; Rosati, Rachele; Yang, Wei; Chen, Vivian S; Demidenko, Eugene; Morra, Rocco P; Miller, Todd W

    2017-06-01

    Purpose: Determine the roles of the PI3K isoforms p110α and p110β in PTEN-deficient, estrogen receptor α (ER)-positive breast cancer, and the therapeutic potential of isoform-selective inhibitors.Experimental Design: Anti-estrogen-sensitive and -resistant PTEN-deficient, ER(+) human breast cancer cell lines, and mice bearing anti-estrogen-resistant xenografts were treated with the anti-estrogen fulvestrant, the p110α inhibitor BYL719, the p110β inhibitor GSK2636771, or combinations. Temporal response to growth factor receptor-initiated signaling, growth, apoptosis, predictive biomarkers, and tumor volumes were measured.Results: p110β primed cells for response to growth factor stimulation. Although p110β inhibition suppressed cell and tumor growth, dual targeting of p110α/β enhanced apoptosis and provided sustained tumor response. The growth of anti-estrogen-sensitive cells was inhibited by fulvestrant, but fulvestrant inconsistently provided additional therapeutic effects beyond PI3K inhibition alone. Treatment-induced decreases in phosphorylation of AKT and Rb were predictive of therapeutic response. Short-term drug treatment induced tumor cell apoptosis and proliferative arrest to induce tumor regression, whereas long-term treatment only suppressed proliferation to provide durable regression.Conclusions: p110β is the dominant PI3K isoform in PTEN-deficient, ER(+) breast cancer cells. Upon p110β inhibition, p110α did not induce significant reactivation of AKT, but combined targeting of p110α/β most effectively induced apoptosis in vitro and in vivo and provided durable tumor regression. Because apoptosis and tumor regression occurred early but not late in the treatment course, and proliferative arrest was maintained throughout treatment, p110α/β inhibitors may be considered short-term cytotoxic agents and long-term cytostatic agents. Clin Cancer Res; 23(11); 2795-805. ©2016 AACR. ©2016 American Association for Cancer Research.

  19. Nurse turnover: a literature review.

    PubMed

    Hayes, Laureen J; O'Brien-Pallas, Linda; Duffield, Christine; Shamian, Judith; Buchan, James; Hughes, Frances; Spence Laschinger, Heather K; North, Nicola; Stone, Patricia W

    2006-02-01

    Ongoing instability in the nursing workforce is raising questions globally about the issue of nurse turnover. A comprehensive literature review was undertaken to examine the current state of knowledge about the scope of the nurse turnover problem, definitions of turnover, factors considered to be determinants of nurse turnover, turnover costs and the impact of turnover on patient, and nurse and system outcomes. Much of the research to date has focused on turnover determinants, and recent studies have provided cost estimations at the organizational level. Further research is needed to examine the impact of turnover on health system cost, and how nurse turnover influences patient and nurse outcomes.

  20. Coated vesicles contain a phosphatidylinositol kinase.

    PubMed

    Campbell, C R; Fishman, J B; Fine, R E

    1985-09-15

    When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.

  1. Mechanism of substrate specificity of phosphatidylinositol phosphate kinases

    PubMed Central

    Muftuoglu, Yagmur; Xue, Yi; Gao, Xiang; Wu, Dianqing; Ha, Ya

    2016-01-01

    The phosphatidylinositol phosphate kinase (PIPK) family of enzymes is primarily responsible for converting singly phosphorylated phosphatidylinositol derivatives to phosphatidylinositol bisphosphates. As such, these kinases are central to many signaling and membrane trafficking processes in the eukaryotic cell. The three types of phosphatidylinositol phosphate kinases are homologous in sequence but differ in catalytic activities and biological functions. Type I and type II kinases generate phosphatidylinositol 4,5-bisphosphate from phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, respectively, whereas the type III kinase produces phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3-phosphate. Based on crystallographic analysis of the zebrafish type I kinase PIP5Kα, we identified a structural motif unique to the kinase family that serves to recognize the monophosphate on the substrate. Our data indicate that the complex pattern of substrate recognition and phosphorylation results from the interplay between the monophosphate binding site and the specificity loop: the specificity loop functions to recognize different orientations of the inositol ring, whereas residues flanking the phosphate binding Arg244 determine whether phosphatidylinositol 3-phosphate is exclusively bound and phosphorylated at the 5-position. This work provides a thorough picture of how PIPKs achieve their exquisite substrate specificity. PMID:27439870

  2. The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways.

    PubMed

    Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T; Portell, Craig A; Lannutti, Brian J; Almasan, Alexandru; Hsi, Eric D

    2013-10-01

    Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

  3. Prolyl isomerase Pin1 promotes amyloid precursor protein (APP) turnover by inhibiting glycogen synthase kinase-3β (GSK3β) activity: novel mechanism for Pin1 to protect against Alzheimer disease.

    PubMed

    Ma, Suk Ling; Pastorino, Lucia; Zhou, Xiao Zhen; Lu, Kun Ping

    2012-03-02

    Alzheimer disease (AD) is characterized by the presence of senile plaques of amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) and neurofibrillary tangles made of hyperphosphorylated Tau. Increasing APP gene dosage or expression has been shown to cause familial early-onset AD. However, whether and how protein stability of APP is regulated is unclear. The prolyl isomerase Pin1 and glycogen synthase kinase-3β (GSK3β) have been shown to have the opposite effects on APP processing and Tau hyperphosphorylation, relevant to the pathogenesis of AD. However, nothing is known about their relationship. In this study, we found that Pin1 binds to the pT330-P motif in GSK3β to inhibit its kinase activity. Furthermore, Pin1 promotes protein turnover of APP by inhibiting GSK3β activity. A point mutation either at Thr-330, the Pin1-binding site in GSK3β, or at Thr-668, the GSK3β phosphorylation site in APP, abolished the regulation of GSK3β activity, Thr-668 phosphorylation, and APP stability by Pin1, resulting in reduced non-amyloidogenic APP processing and increased APP levels. These results uncover a novel role of Pin1 in inhibiting GSK3β kinase activity to reduce APP protein levels, providing a previously unrecognized mechanism by which Pin1 protects against Alzheimer disease.

  4. 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.

  5. Inhibitory effects of ethanol on phosphatidylinositol breakdown in pancreatic acini

    SciTech Connect

    Towner, S.J.; Peppin, J.F.; Tsukamoto, H.

    1986-03-01

    Recently the physiological relationship between the phospholipid effect and secretagogue-induced cellular function has begun to be understood. In this study, the authors investigated acute and chronic effects of ethanol on phosphatidylinositol (PI) synthesis and breakdown in pancreatic acini. Five pairs of male Wistar rats were intragastrically infused for 30 days with high fat diet (25% total calories) plus ethanol or isocaloric dextrose. After intoxication, isolated in HEPES media, followed by 30 min incubation with CCK-8 (0, 100, 300 or 600 pM) and ethanol (0 or 100 mM). Acinar lipids were extracted and counted for labeled PI. Incorporation of /sup 3/H-inositol into alcoholic acinar PI was reduced to 38.2% of that in controls. A percent maximal PI break down by CCK-8 was similar in the two groups (13-24% of basal). However, the magnitude of PI breakdown was markedly lower in alcoholic acini (482 vs 1081 dpm) due to the decreased PI synthesis rate. The presence of 100 mM ethanol in the media further inhibited the breakdown by 50% in this group. These results strongly indicate that chronic ethanol intoxication inhibits PI synthesis and breakdown in pancreatic acini, and that this inhibition can be potentiated by acute ethanol administration.

  6. Endosomal Phosphatidylinositol 3-Kinase Is Essential for Canonical GPCR Signaling.

    PubMed

    Uchida, Yasunori; Rutaganira, Florentine U; Jullié, Damien; Shokat, Kevan M; von Zastrow, Mark

    2017-01-01

    G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the β2-adrenoceptor (β2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by β2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.

  7. Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: Effects of a cyclic AMP phosphodiesterase inhibitor

    SciTech Connect

    Homa, S.T.; Webster, S.D.; Russell, R.K. )

    1991-08-01

    The turnover of (32P)orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in (32P)phosphatidic acid (PA) and an increase in (32P)-phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in (32P)PC and (32P)phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in (32P)PC and (32P)PE which accompanied GVBD. The increase in (32P)phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid.

  8. Phosphatidylinositol kinase activities in Trypanosoma cruzi epimastigotes.

    PubMed

    Gimenez, Alba Marina; Gesumaría, María Celeste; Schoijet, Alejandra C; Alonso, Guillermo D; Flawiá, Mirtha M; Racagni, Graciela E; Machado, Estela E

    2015-01-01

    Phosphatidylinositol (PtdIns) metabolism through phosphatidylinositol kinase (PIKs) activities plays a central role in different signaling pathways. In Trypanosoma cruzi, causative agent of Chagas disease, PIKs have been proposed as target for drug design in order to combat this pathogen. In this work, we studied the classes of PI4K, PIPK and PI3K that could participate in signaling pathways in T. cruzi epimastigote forms. For this reason, we analyzed their enzymatic parameters and detailed responses to avowed kinase inhibitors (adenosine, sodium deoxycholate, wortmannin and LY294002) and activators (Ca(2+), phosphatidic acid, spermine and heparin). Our results suggest the presence and activity of a class III PI4K, a class I PIPK, a class III PI3K previously described (TcVps34) and a class I PI3K. Class I PI3K enzyme, here named TcPI3K, was cloned and expressed in a bacterial system, and their product was tested for kinase activity. The possible participation of TcPI3K in central cellular events of the parasite is also discussed.

  9. The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Zhang, Tao

    2011-05-01

    Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl-1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA, activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1, 4, 5-trisphosphate (IP3) but not PKC.

  10. Monocyte-Derived Interleukin 1: Effects on Aortic Contraction and Phosphatidylinositol Turnover

    DTIC Science & Technology

    1988-11-29

    TELEPHONE (Include Area Code) 2c. OFFICE SYMBOL Regina E. Hunt, Command Editor (202) 295-0198 NSB/ADMIN/NMRI DD FORM 1473, 84 MAR 83 APR edition may be...REPORT NUMBER(S) S MONITORING ORGANIZAflON REPORT NUMBER(S) K"Val 88-96 6a. NJAME OF PERFORMING ORGANIZAr;ON 6o OFFICE SYMBOL 7a. NJAME OF MONITORING...SPONSORING 8b, OFFICE SYMBOL 9 PROCUREMENT iNSTRUMENT IDENTIFICATION NUMBER ORGANIZATION Naval Medical- (if applicable) Research & Development CommiandI 8C

  11. Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

    PubMed Central

    Kirk, C J; Michell, R H; Hems, D A

    1981-01-01

    In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution. PMID:7030316

  12. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

  13. Avifauna: Turnover on Islands.

    PubMed

    Mayr, E

    1965-12-17

    The percentage of endemic species of birds on islands increases with island area at a double logarithmic rate. This relation is apparently due to extinction, which is more rapid the smaller the island. The turnover resulting from extinction and replacement appears to be far more rapid than hitherto suspected.

  14. Transcriptional analysis of three major putative phosphatidylinositol kinase genes in a parasitic protozoan, Giardia lamblia.

    PubMed

    Hernandez, Yunuen; Zamora, Gus; Ray, Suparna; Chapoy, Jaime; Chavez, Edna; Valvarde, Robert; Williams, Ebonye; Aley, Stephen B; Das, Siddhartha

    2007-01-01

    The current investigation evaluates the expression of phosphatidylinositol kinase (PIK) genes in the parasitic protozoan, Giardia lamblia. The G. lamblia Genome Database revealed the presence of two putative phosphatidylinositol-3-kinase (gPI3K) and one phosphatidylinositol-4-kinase (gPI4K) genes resembling the catalytic subunit of eukaryotic PIKs. Primers, designed to amplify mRNA of these three genes, were used to measure transcription by quantitative reverse-transcriptase polymerase chain reactions. Results suggest that all three PIK genes are expressed in non-encysting and encysting trophozoites. The relative levels of the mRNA were highest in parasites cultured in pre-encysting medium that contained no bile. Two inhibitors of PI3K, LY 294002 and wortmannin were found to inhibit the growth of the trophozoite in culture. However, wortmannin was more effective than LY294002. Altogether, the present study indicates that Giardia is capable of expressing PIKs that are necessary for the growth and differentiation of this pathogen.

  15. Phosphatidylinositol 3-Kinase and Antiestrogen Resistance in Breast Cancer

    PubMed Central

    Miller, Todd W.; Balko, Justin M.; Arteaga, Carlos L.

    2011-01-01

    Although antiestrogen therapies targeting estrogen receptor (ER) α signaling prevent disease recurrence in the majority of patients with hormone-dependent breast cancer, a significant fraction of patients exhibit de novo or acquired resistance. Currently, the only accepted mechanism linked with endocrine resistance is amplification or overexpression of the ERBB2 (human epidermal growth factor receptor 2 [HER2]) proto-oncogene. Experimental and clinical evidence suggests that hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway, the most frequently mutated pathway in breast cancer, promotes antiestrogen resistance. PI3K is a major signaling hub downstream of HER2 and other receptor tyrosine kinases. PI3K activates several molecules involved in cell-cycle progression and survival, and in ER-positive breast cancer cells, it promotes estrogen-dependent and -independent ER transcriptional activity. Preclinical tumor models of antiestrogen-resistant breast cancer often remain sensitive to estrogens and PI3K inhibition, suggesting that simultaneous targeting of the PI3K and ER pathways may be most effective. Herein, we review alterations in the PI3K pathway associated with resistance to endocrine therapy, the state of clinical development of PI3K inhibitors, and strategies for the clinical investigation of such drugs in hormone receptor–positive breast cancer. PMID:22010023

  16. Predicting Turnover Intentions and Turnover Behavior: A Multivariate Analysis.

    ERIC Educational Resources Information Center

    Parasuraman, Saroj

    1982-01-01

    Assessed the relative influence of personal, attitudinal, and behavioral variables on behavioral intentions and voluntary turnover among nonsupervisory plant workers. Results show that personal variables have little direct effect on turnover; rather, their influence on turnover is channeled through their effects on behavioral intentions. (Author)

  17. Phosphatidylinositol 4-phosphate 5-kinase α and Vav1 mutual cooperation in CD28-mediated actin remodeling and signaling functions.

    PubMed

    Muscolini, Michela; Camperio, Cristina; Porciello, Nicla; Caristi, Silvana; Capuano, Cristina; Viola, Antonella; Galandrini, Ricciarda; Tuosto, Loretta

    2015-02-01

    Phosphatidylinositol 4,5-biphosphate (PIP2) is a cell membrane phosphoinositide crucial for cell signaling and activation. Indeed, PIP2 is a pivotal source for second messenger generation and controlling the activity of several proteins regulating cytoskeleton reorganization. Despite its critical role in T cell activation, the molecular mechanisms regulating PIP2 turnover remain largely unknown. In human primary CD4(+) T lymphocytes, we have recently demonstrated that CD28 costimulatory receptor is crucial for regulating PIP2 turnover by allowing the recruitment and activation of the lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5Kα). We also identified PIP5Kα as a key modulator of CD28 costimulatory signals leading to the efficient T cell activation. In this study, we extend these data by demonstrating that PIP5Kα recruitment and activation is essential for CD28-mediated cytoskeleton rearrangement necessary for organizing a complete signaling compartment leading to downstream signaling functions. We also identified Vav1 as the linker molecule that couples the C-terminal proline-rich motif of CD28 to the recruitment and activation of PIP5Kα, which in turn cooperates with Vav1 in regulating actin polymerization and CD28 signaling functions. Copyright © 2015 by The American Association of Immunologists, Inc.

  18. Phosphatidylinositol 4-Phosphate Negatively Regulates Chloroplast Division in Arabidopsis[OPEN

    PubMed Central

    Okazaki, Kumiko; Miyagishima, Shin-ya; Wada, Hajime

    2015-01-01

    Chloroplast division is performed by the constriction of envelope membranes at the division site. Although constriction of a ring-like protein complex has been shown to be involved in chloroplast division, it remains unknown how membrane lipids participate in the process. Here, we show that phosphoinositides with unknown function in envelope membranes are involved in the regulation of chloroplast division in Arabidopsis thaliana. PLASTID DIVISION1 (PDV1) and PDV2 proteins interacted specifically with phosphatidylinositol 4-phosphate (PI4P). Inhibition of phosphatidylinositol 4-kinase (PI4K) decreased the level of PI4P in chloroplasts and accelerated chloroplast division. Knockout of PI4Kβ2 expression or downregulation of PI4Kα1 expression resulted in decreased levels of PI4P in chloroplasts and increased chloroplast numbers. PI4Kα1 is the main contributor to PI4P synthesis in chloroplasts, and the effect of PI4K inhibition was largely abolished in the pdv1 mutant. Overexpression of DYNAMIN-RELATED PROTEIN5B (DRP5B), another component of the chloroplast division machinery, which is recruited to chloroplasts by PDV1 and PDV2, enhanced the effect of PI4K inhibition, whereas overexpression of PDV1 and PDV2 had additive effects. The amount of DRP5B that associated with chloroplasts increased upon PI4K inhibition. These findings suggest that PI4P is a regulator of chloroplast division in a PDV1- and DRP5B-dependent manner. PMID:25736058

  19. Phosphatidylinositol synthase and phosphatidylinositol/inositol exchange reactions in turkey erythrocyte membranes.

    PubMed Central

    McPhee, F; Lowe, G; Vaziri, C; Downes, C P

    1991-01-01

    Unlike human erythrocytes, those from avian species, such as turkeys and chicks, rapidly incorporate myo-[3H]inositol into membrane phospholipids. The mechanisms regulating [3H]Ins labelling of phosphatidylinositol have been investigated using turkey erythrocyte membranes. In the absence of added nucleotides, [3H]inositol incorporation appears to proceed via phosphatidylinositol/inositol exchange, with a Km for inositol of 0.01 mM. The reaction was dependent upon divalent cations, either Mg2+ or Mn2+, with the latter metal ion being the more effective. [3H]Inositol incorporation was accelerated by CMP, especially when the concentration of Ins was greater than the Km for the exchange reaction. CMP-dependent labelling of PtdIns had a Km for inositol of 0.3 mM and for CMP of 0.015 mM. Divalent cations were also required for this reaction: activity peaked at 0.5 mM-Mn2+ and declined at higher concentrations. At relatively high concentrations, Mg2+ was more effective than Mn2+, with peak activity being achieved above 10 mM. CMP-dependent incorporation of [3H]inositol appears to reflect an exchange reaction catalysed by PtdIns synthase. Definitive evidence for the occurrence of PtdIns synthase in turkey erythrocyte membranes was obtained by demonstrating the formation of [14C]CMP-phosphatidate from [14C]CMP. The radioactivity could be efficiently chased from [14C]CMP-phosphatidate in the presence of unlabelled inositol. The detection of PtdIns synthase activity in morphologically simple turkey erythrocytes should help to clarify the subcellular distribution of this important component of the phosphatidylinositol cycle. PMID:1850237

  20. Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.

    PubMed

    Posor, York; Eichhorn-Gruenig, Marielle; Puchkov, Dmytro; Schöneberg, Johannes; Ullrich, Alexander; Lampe, André; Müller, Rainer; Zarbakhsh, Sirus; Gulluni, Federico; Hirsch, Emilio; Krauss, Michael; Schultz, Carsten; Schmoranzer, Jan; Noé, Frank; Haucke, Volker

    2013-07-11

    Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.

  1. Severe low turnover osteoporosis.

    PubMed

    Pietrogrande, Luca

    2007-08-01

    Severe osteoporosis, a situation with fractures, can worsen in the case of poor response to usual therapies, such as bisphosphonates associated with calcium and vitamin D, especially if bone turnover is strongly suppressed. One way of inverting the poor evolution of non-responders is to use Teriparatide. The case of a non-responder is reported, with considerations about the possibility of detecting these patients before a new fracture takes place.

  2. Turnover of Junior Officers

    DTIC Science & Technology

    1978-09-01

    by reference to Maslow’s Hierarchy of Needs theory (24:42). The lowest needs on Maslow’s hierarchy are for survival and security. Pay is the principal... Hierarchy of Needs Theory (2:42) suggests that once pay is adequate to satisfy the lower needs for survival and security, it decreases in importance to...satisfaction increases. This result indicated that pay does not play the role depicted in the synthesized turnover model in Chapter II. Maslow’s

  3. Alpha/sub 1/ receptor stimulated phosphatidylinositol hydrolysis in rat cerebral cortex

    SciTech Connect

    Raulli, R.; Crews, F.T.

    1986-03-05

    The potency of various alpha adrenergic compounds on stimulation of phosphatidylinositol (PI) hydrolysis was determined using (/sup 3/H)-inositol labelled cerebral cortical slices. Norepinephrine-induced PI hydrolysis was inhibited by the alpha/sub 1/ selective antagonist prazosin (1 ..mu..M) but not the beta receptor antagonist propranolol (1 ..mu..M). Tramazoline, (-)-ephedrine, and (+/-)-phenylpropanolamine were all found to be partial agonists at 1 mM concentrations. Clonidine, naphazoline, trazodone, and the novel antidepressant mianserin at concentrations of 100 ..mu..M to 1 mM produced no significant increase in PI hydrolysis above control levels. The relationship between responses and receptor binding will be discussed.

  4. [Experiences of nurse turnover].

    PubMed

    Lee, Yun-Jung; Kim, Kwuy-Bun

    2008-04-01

    This study was designed to search for nursing intervention strategies centering around the meaning structure of the nurse's turnover experience by applying phenomenological methods. The participants were 6 nurses in small and medium sized hospitals who had experienced at least 1 turnover. Data were collected used MP3 records. The data analysis was done by Giorgi (1985) method. The results were divided into the following categories: 1) Careless decision: wrong decisions, imprudent desire, insufficient patience, unclear future, 2) Inappropriate working environment: irregular working hours, high workload, poor working environment, insufficient understanding of related divisions, lack of opinion collection, low salary, 3) Interpersonal relations problems: discord with colleagues, difficulty in relationships with others, difficulty in daily lives, 4) Lack of specialization: feeling of inertia, lack of role identification, lack of self identification, 5) Inappropriate coping: regret with clinical challenges, difficulty with a new environment, repentance, expectation, relative humility, 6) New self-dignity: expectation, new challenge, relaxing lives, decisions based on future-oriented confidence. The finding of this study will offer profound information on the nurse's turnover experience and provide basic raw materials for improving the quality of nursing performance and contribute to the development of hospital organization.

  5. Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

    SciTech Connect

    Smith, Adrian L.; D’Angelo, Noel D.; Bo, Yunxin Y.; Booker, Shon K.; Cee, Victor J.; Herberich, Brad; Hong, Fang-Tsao; Jackson, Claire L.M.; Lanman, Brian A.; Liu, Longbin; Nishimura, Nobuko; Pettus, Liping H.; Reed, Anthony B.; Tadesse, Seifu; Tamayo, Nuria A.; Wurz, Ryan P.; Yang, Kevin; Andrews, Kristin L.; Whittington, Douglas A.; McCarter, John D.; Miguel, Tisha San; Zalameda, Leeanne; Jiang, Jian; Subramanian, Raju; Mullady, Erin L.; Caenepeel, Sean; Freeman, Daniel J.; Wang, Ling; Zhang, Nancy; Wu, Tian; Hughes, Paul E.; Norman, Mark H.

    2012-09-17

    A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

  6. Inositol induces a profound alteration in the pattern and rate of synthesis and turnover of membrane lipids in Saccharomyces cerevisiae.

    PubMed

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

    2006-08-11

    The addition of inositol to actively growing yeast cultures causes a rapid increase in the rate of synthesis of phosphatidylinositol and, simultaneously, triggers changes in the expression of hundreds of genes. We now demonstrate that the addition of inositol to yeast cells growing in the presence of choline leads to a dramatic reprogramming of cellular lipid synthesis and turnover. The response to inositol includes a 5-6-fold increase in cellular phosphatidylinositol content within a period of 30 min. The increase in phosphatidylinositol content appears to be dependent upon fatty acid synthesis. Phosphatidylcholine turnover increased rapidly following inositol addition, a response that requires the participation of Nte1p, an endoplasmic reticulum-localized phospholipase B. Mass spectrometry revealed that the acyl species composition of phosphatidylinositol is relatively constant regardless of supplementation with inositol or choline, whereas phosphatidylcholine acyl species composition is influenced by both inositol and choline. In medium containing inositol, but lacking choline, high levels of dimyristoylphosphatidylcholine were detected. Within 60 min following the addition of inositol, dimyristoylphosphatidylcholine levels had decreased from approximately 40% of total phosphatidylcholine to a basal level of less than 5%. nte1Delta cells grown in the absence of inositol and in the presence of choline exhibited lower levels of dimyristoylphosphatidylcholine than wild type cells grown under these same conditions, but these levels remained largely constant after the addition of inositol. These results are discussed in relationship to transcriptional regulation known to be linked to lipid metabolism in yeast.

  7. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    PubMed

    Robinson, M L; Carman, G M

    1982-01-01

    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  8. Long noncoding RNA turnover

    PubMed Central

    Yoon, Je-Hyun; Kim, Jiyoung; Gorospe, Myriam

    2015-01-01

    Most RNAs transcribed in mammalian cells lack protein-coding sequences. Among them is a vast family of long (>200 nt) noncoding (lnc)RNAs. LncRNAs can modulate cellular protein expression patterns by influencing the transcription of many genes, the post-transcriptional fate of mRNAs and ncRNAs, and the turnover and localization of proteins. Given the broad impact of lncRNAs on gene regulation, there is escalating interest in elucidating the mechanisms that govern the steady-state levels of lncRNAs. In this review, we summarize our current knowledge of the factors and mechanisms that modulate mammalian lncRNA stability. PMID:25769416

  9. Biochemical and biological functions of class I phosphatidylinositol transfer proteins.

    PubMed

    Cockcroft, Shamshad; Carvou, Nicolas

    2007-06-01

    Phosphoinositides function in a diverse array of cellular activities. They include a role as substrate for lipid kinases and phospholipases to generate second messengers, regulators of the cytoskeleton, of enzymes and of ion channels, and docking sites for reversible recruitment of proteins to membranes. Mammalian phosphatidylinositol transfer proteins, PITPalpha and PITPbeta are paralogs that share 77% sequence identity and contain a hydrophobic cavity that can sequester either phosphatidylinositol or phosphatidylcholine. A string of 11 amino acid residues at the C-terminal acts as a "lid" which shields the lipid from the aqueous environment. PITPs in vitro can facilitate inter-membrane lipid transfer and this requires the movement of the "lid" to allow the lipid cargo to be released. Thus PITPs are structurally designed for delivering lipid cargo and could thus participate in cellular events that are dependent on phosphatidylinositol or derivatives of phosphatidylinositol. Phosphatidylinositol, the precursor for all phosphoinositides is synthesised at the endoplasmic reticulum and its distribution to other organelles could be facilitated by PITPs. Here we highlight recent studies that report on the three-dimensional structures of the different PITP forms and suggest how PITPs are likely to dock at the membrane surface for lipid delivery and extraction. Additionally we discuss whether PITPs are important regulators of sphingomyelin metabolism, and finally describe recent studies that link the association of PITPs with diverse functions including membrane traffic at the Golgi, neurite outgrowth, cytokinesis and stem cell growth.

  10. RNA turnover in Trypanosoma brucei.

    PubMed Central

    Ehlers, B; Czichos, J; Overath, P

    1987-01-01

    Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA. Images PMID:2436040

  11. Metabolic turnover of myelin glycerophospholipids.

    PubMed

    Morell, P; Ousley, A H

    1994-08-01

    The apparent half life for metabolic turnover of glycerophospholipids in the myelin sheath, as determined by measuring the rate of loss of label in a myelin glycerophospholipid following radioactive precursor injection, varies with the radioactive precursor used, age of animal, and time after injection during which metabolic turnover is studied. Experimental strategies for resolving apparent inconsistencies consequent to these variables are discussed. Illustrative data concerning turnover of phosphatidylcholine (PC) in myelin of rat brain are presented. PC of the myelin membrane exhibits heterogeneity with respect to metabolic turnover rates. There are at least two metabolic pools of PC in myelin, one with a half life of the order of days, and another with a half life of the order of weeks. To a significant extent biphasic turnover is due to differential turnover of individual molecular species (which differ in acyl chain composition). The two predominant molecular species of myelin PC turnover at very different rates (16:0, 18:1 PC turning over several times more rapidly than 18:0, 18:1 PC). Therefore, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Possible mechanisms for differential turnover of molecular species are discussed, as are other factors that may contribute to a multiphasic turnover of glycerophospholipids.

  12. 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.

  13. Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts.

    PubMed

    Hata, Jonathan A; Petrofski, Jason A; Schroder, Jacob N; Williams, Matthew L; Timberlake, Sarah H; Pippen, Anne; Corwin, Michael T; Solan, Amy K; Jakoi, Andre; Gehrig, Thomas R; Kontos, Christopher D; Milano, Carmelo A

    2005-06-01

    Fifty percent of human aortocoronary saphenous vein grafts are occluded after 10 years. Intimal hyperplasia is an initial step in graft occlusion and consists of vascular smooth muscle cell proliferation. Phosphatidylinositol 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), are important regulators of vascular smooth muscle cell proliferation, migration, and cell death. This study tests whether overexpression of PTEN in aortocoronary saphenous vein grafts can reduce intimal hyperplasia. Adult dogs underwent aortocoronary bypass grafting to the left anterior descending artery by using the autologous saphenous vein. Saphenous vein grafts were treated with phosphate-buffered saline (n = 9), empty adenovirus (n = 8), or adenovirus encoding for PTEN (n = 8). Arteriography at 30 and 90 days assessed saphenous vein graft patency. A subset received saphenous vein grafts treated with a marker transgene (beta-galactosidase, n = 3), empty adenovirus (n = 4), or adenovirus encoding for PTEN (n = 4) and were killed on postoperative day 3 to confirm expression. Vascular smooth muscle cells were isolated from canine saphenous vein infected with adenovirus encoding for PTEN, and immunoblotting and proliferation assays were performed. Saphenous vein graft transgene expression was confirmed by means of immunohistochemistry, immunoblotting, and polymerase chain reaction. Arteriograms revealed all saphenous vein grafts to be patent. Saphenous vein grafts treated with adenovirus encoding for PTEN demonstrated reduced intimal area compared with those treated with empty adenovirus and phosphate-buffered saline (1.39 +/- 0.11 vs 2.35 +/- 0.3 and 2.57 +/- 0.4 mm 2 , P < .05), and the intima/media ratio was lower in saphenous vein grafts treated with adenovirus encoding for PTEN (0.50 +/- 0.05 vs 1.43 +/- 0.18 and 1.11 +/- 0.14, P < .005). PTEN overexpression in vascular smooth muscle cells inhibited platelet

  14. Integrating Turnover Reasons and Shocks with Turnover Decision Processes

    ERIC Educational Resources Information Center

    Maertz, Carl P., Jr.; Kmitta, Kayla R.

    2012-01-01

    We interviewed and classified 186 quitters from many jobs and organizations via a theoretically-based protocol into five decision process types. We then tested exploratory hypotheses comparing users of these types on their propensity to report certain turnover reasons and turnover shocks. "Impulsive-type quitters," with neither a job offer in hand…

  15. Integrating Turnover Reasons and Shocks with Turnover Decision Processes

    ERIC Educational Resources Information Center

    Maertz, Carl P., Jr.; Kmitta, Kayla R.

    2012-01-01

    We interviewed and classified 186 quitters from many jobs and organizations via a theoretically-based protocol into five decision process types. We then tested exploratory hypotheses comparing users of these types on their propensity to report certain turnover reasons and turnover shocks. "Impulsive-type quitters," with neither a job offer in hand…

  16. Mitochondrial biogenesis and turnover.

    PubMed

    Diaz, Francisca; Moraes, Carlos T

    2008-07-01

    Mitochondrial biogenesis is a complex process involving the coordinated expression of mitochondrial and nuclear genes, the import of the products of the latter into the organelle and turnover. The mechanisms associated with these events have been intensively studied in the last 20 years and our understanding of their details is much improved. Mitochondrial biogenesis requires the participation of calcium signaling that activates a series of calcium-dependent protein kinases that in turn activate transcription factors and coactivators such as PGC-1alpha that regulates the expression of genes coding for mitochondrial components. In addition, mitochondrial biogenesis involves the balance of mitochondrial fission-fusion. Mitochondrial malfunction or defects in any of the many pathways involved in mitochondrial biogenesis can lead to degenerative diseases and possibly play an important part in aging.

  17. Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate

    SciTech Connect

    Lee, Myungho; Bell, R.M. )

    1991-01-29

    The mechanism of protein kinase C (PKC) activation by phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI) was investigated by using Triton X-100 mixed micellar methods. The activation of PKC by PIP{sub 2}, for which maximal activity was 60% of that elicited by sn-1,2-diacylglycerol (DAG), was similar to activation by DAG in several respects: (1) activation by PIP{sub 2} and DAG required phosphatidylserine (PS) as a phospholipid cofactor, (2) PIP{sub 2} and DAG reduced the concentration of Ca{sup 2+} and PS required for activation, (3) the concentration dependences of activation by PIP{sub 2} and DAG depended on the concentration of PS, and (4) PIP{sub 2} and DAG complemented one another to achieve maximal activation. On the other hand, PIP{sub 2} activation of the PKC differed from activation by DAG in several respects. With increasing concentrations of PIP{sub 2}, (1) the optimal concentration of PS required was constant at 12 mol%, (2) the maximal activity at 12 mol% PS increased, and (3) the cooperativity for PS decreased. PIP{sub 2} did not inhibit ({sup 3}H)phorbol 12,13-dibutyrate (PDBu) binding of PKC at saturating levels of PS; however, at subsaturating levels of PS, PIP{sub 2} enhanced ({sup 3}H)PDBu binding by acting as a phospholipid cofactor. PIP did not function as an activator but served as a phospholipid cofactor in the presence of PS. These data establish that PIP{sub 2}, PIP, and PI can function to spare, in part, the PS phospholipid cofactor requirement of PKC, and they demonstrate that PIP{sub 2} but not PIP and PI can function as a lipid activator of PKC by mechanisms distinct from those of DAG and phorbol esters.

  18. Commitment Profiles and Employee Turnover

    ERIC Educational Resources Information Center

    Stanley, Laura; Vandenberghe, Christian; Vandenberg, Robert; Bentein, Kathleen

    2013-01-01

    We examined how affective (AC), normative (NC), perceived sacrifice (PS), and few alternatives (FA) commitments combine to form profiles and determine turnover intention and turnover. We theorized that three mechanisms account for how profiles operate, i.e., the degree to which membership is internally regulated, the perceived desirability and…

  19. Salary, Performance, and Superintendent Turnover

    ERIC Educational Resources Information Center

    Grissom, Jason A.; Mitani, Hajime

    2016-01-01

    Purpose: Superintendent retention is an important goal for many school districts, yet the factors contributing to superintendent turnover are poorly understood. Most prior quantitative studies of superintendent turnover have relied on small, cross-sectional samples, limiting the evidence base. Utilizing longitudinal administrative records from…

  20. Teacher Turnover: A Conceptual Analysis

    ERIC Educational Resources Information Center

    Martinez-Garcia, Cynthia; Slate, John R.

    2009-01-01

    In this article we reviewed the available literature concerning teacher turnover. The seriousness of this issue was addressed as cause for concern is clearly present. Issues we examined in this conceptual analysis were the federal government's role in public education, the No Child Left Behind Act, teacher turnover, teacher retention, teacher…

  1. Salary, Performance, and Superintendent Turnover

    ERIC Educational Resources Information Center

    Grissom, Jason A.; Mitani, Hajime

    2016-01-01

    Purpose: Superintendent retention is an important goal for many school districts, yet the factors contributing to superintendent turnover are poorly understood. Most prior quantitative studies of superintendent turnover have relied on small, cross-sectional samples, limiting the evidence base. Utilizing longitudinal administrative records from…

  2. Insulin-stimulated phosphatidylinositol 3-kinase activity and 2-deoxy-D-glucose uptake in rat skeletal muscles.

    PubMed

    Elmendorf, J S; Damrau-Abney, A; Smith, T R; David, T S; Turinsky, J

    1995-03-28

    To date there is suggestive evidence that phosphatidylinositol 3-kinase participates in insulin-stimulated glucose transport. However, its involvement in skeletal muscle, a major site of insulin-stimulated glucose disposal, has not been addressed. Therefore, we tested the effects of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase, on insulin-stimulated 2-deoxyglucose uptake by rat soleus muscle in vitro: Wortmannin (1 microM) reversibly inhibited insulin-induced 2-deoxyglucose uptake in soleus muscle by 44%. Inclusion of 5 microM wortmannin in the incubation medium completely abolished the insulin-induced increment in 2-deoxyglucose uptake. In conclusion, the insulin-signaling cascade linking insulin-receptor tyrosine kinase activation to glucose uptake in skeletal muscle.

  3. Occupational stress and employee turnover.

    PubMed

    Bridger, Robert S; Day, Andrea J; Morton, Kate

    2013-01-01

    Questionnaire data captured in January-March 2007 were examined in relation to turnover in males and females during the next five years. In general, most of the workplace stressors (such as role conflict or peer support) were not antecedents of turnover in any group. Junior personnel with psychological strain in 2007 had an increased risk of turnover in the next five years. Low commitment to the service in 2007 increased the odds of turnover in male and female juniors and in female officers. Female juniors with less effective skills for coping with stress and who exercised less frequently on a weekly basis were more likely to leave. An incidental finding was that the odds of turnover were three times greater in female officers with children than in female officers with no children. Stress management interventions focusing on effective coping and sports and exercise participation which are targeted appropriately may improve retention.

  4. Work and Career considerations in Understanding Employee Turnover Intentions and Turnover: Development of the Turnover Diagnostic.

    DTIC Science & Technology

    1984-08-01

    researchers have noted that organizations perceived not to link rewards to performance had higher turnover ( Hellriegel & White, 1973; Hulln, 1966, 1968...Telly, French, & Scott, 1971). Similarly, some company policy and administrative Issues related to pay and promotion ( Hellriegel & White, 1973...practices are a strong correlate of turnover (Dansereau, Cashman, & Graen, 1973; Fleishman & Harris, 1962; Graen & Ginsburgh, 1977; Hellriegel & White

  5. A phosphatidylinositol-linkage-deficient T-cell mutant contains insulin-sensitive glycosyl-phosphatidylinositol.

    PubMed Central

    Avila, M A; Clemente, R; Varela-Nieto, I

    1992-01-01

    Glycosyl-phosphatidylinositol molecules, acting as both signal transduction elements and membrane protein anchors, have been proposed to play a role during T-cell activation. The MVB2 cell line is a mutant, derived from the wild-type T-T hybrid YH.16.33, which has a defect in the biosynthesis of PtdIns-protein linkages. As a consequence, MVB2 mutants are defective in activation through the T-cell receptor. Despite the lack of glycosyl-PtdIns anchors in the mutant MVB2 cells, a comparison of the levels and structural features of the insulin-sensitive glycosyl-PtdIns between the MVB2 and YH.16.33 lineages indicates that both cell lines are identical in this respect. The time course for insulin-responsiveness coincides in both cell lines, with maximal hydrolysis 30 s after insulin addition. The ultimate localization of insulin-regulated glycosyl-PtdIns at the outer surface of the cell membrane is also similar. These data indicate that the glycosyl-PtdIns whose hydrolysis is regulated by insulin is not anchoring proteins at the cell surface of T-lymphocytes. Images Fig. 2. PMID:1532490

  6. Phosphatidylinositol-3-kinase as a putative target for anticancer action of clotrimazole.

    PubMed

    Furtado, Cristiane M; Marcondes, Mariah C; Carvalho, Renato S; Sola-Penna, Mauro; Zancan, Patricia

    2015-05-01

    Clotrimazole (CTZ) has been proposed as an antitumoral agent because of its properties that inhibit glycolytic enzymes and detach them from the cytoskeleton. However, the broad effects of the drug, e.g., acting on different enzymes and pathways, indicate that CTZ might also affect several signaling pathways. In this study, we show that CTZ interferes with the human breast cancer cell line MCF-7 after a short incubation period (4 h), thereby diminishing cell viability, promoting apoptosis, depolarizing mitochondria, inhibiting key glycolytic regulatory enzymes, decreasing the intracellular ATP content, and permeating plasma membranes. CTZ treatment also interferes with autophagy. Moreover, when the incubation is performed under hypoxic conditions, certain effects of CTZ are enhanced, such as phosphatidylinositol-3-phosphate kinase (PI3K), which is inhibited upon CTZ treatment; this inhibition is potentiated under hypoxia. CTZ-induced PI3K inhibition is not caused by upstream effects of CTZ because the drug does not affect the interaction of the PI3K regulatory subunit and the insulin receptor substrate (IRS)-1. Additionally, CTZ directly inhibits human purified PI3K in a dose-dependent and reversible manner. Pharmacologic and in silico results suggest that CTZ may bind to the PI3K catalytic site. Therefore, we conclude that PI3K is a novel, putative target for the antitumoral effects of CTZ, interfering with autophagy, apoptosis, cell division and viability.

  7. Phospholipase D specific for the phosphatidylinositol anchor of cell-surface proteins is abundant in plasma

    SciTech Connect

    Low, M.G.; Prasad, A.R.S.

    1988-02-01

    An enzyme activity capable of degrading the glycosyl-phosphatidylinositol membrane anchor of cell-surface proteins has previously been reported in a number of mammalian tissues. The experiments reported here demonstrate that this anchor-degrading activity is also abundant in mammalian plasma. The activity was inhibited by EGTA or 1,10-phenanthroline. It was capable of removing the anchor from alkaline phosphatase, 5'-nucleotidase, and variant surface glycoprotein but had little or no activity toward phosphatidylinositol or phosphatidylcholine. Phosphatidic acid was the only /sup 3/H-labeled product when this enzyme hydrolyzed (/sup 3/H)myristate-labeled variant surface glycoprotein. It could be distinguished from the Ca/sup 2/=-dependent inositol phospholipid-specific phospholipase C activity in several rat tissues on the basis of its molecular size and its sensitivity to 1,10-phenanthroline. The data therefore suggest that this activity is due to a phospholipase D with specificity for glycosylphosphatidylinositol structures. Although the precise physiological function of this anchor-specific phospholipase D remains to be determined, these findings indicate that it could play an important role in regulating the expression and release of cell-surface proteins in vivo.

  8. Tyrosine phosphorylation of type Igamma phosphatidylinositol phosphate kinase by Src regulates an integrin-talin switch.

    PubMed

    Ling, Kun; Doughman, Renee L; Iyer, Vidhya V; Firestone, Ari J; Bairstow, Shawn F; Mosher, Deane F; Schaller, Michael D; Anderson, Richard A

    2003-12-22

    Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIgamma661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIgamma661 and Src. The phosphorylation of Y644 results in an approximately 15-fold increase in binding affinity to the talin head domain and blocks beta-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a "molecular switch" for talin binding between PIPKIgamma661 and beta-integrin that may regulate dynamic FA turnover.

  9. Phosphatidylinositol 5-phosphate 4-kinase regulates early endosomal dynamics during clathrin-mediated endocytosis

    PubMed Central

    2017-01-01

    ABSTRACT Endocytic turnover is essential for the regulation of the protein composition and function of the plasma membrane, and thus affects the plasma membrane levels of many receptors. In Drosophila melanogaster photoreceptors, photon absorption by the G-protein-coupled receptor (GPCR) rhodopsin 1 (Rh1; also known as NinaE) triggers its endocytosis through clathrin-mediated endocytosis (CME). We find that CME of Rh1 is regulated by phosphatidylinositol 5 phosphate 4-kinase (PIP4K). Flies lacking PIP4K show mislocalization of Rh1 on expanded endomembranes within the cell body. This mislocalization of Rh1 was dependent on the formation of an expanded Rab5-positive compartment. The Rh1-trafficking defect in PIP4K-depleted cells could be suppressed by downregulating Rab5 function or by selectively reconstituting PIP4K in the PI3P-enriched early endosomal compartment of photoreceptors. We also found that loss of PIP4K was associated with increased CME and an enlarged Rab5-positive compartment in cultured Drosophila cells. Collectively, our findings define PIP4K as a novel regulator of early endosomal homeostasis during CME. PMID:28507272

  10. Phosphatidylinositol 5-phosphate 4-kinase regulates early endosomal dynamics during clathrin-mediated endocytosis.

    PubMed

    Kamalesh, Kumari; Trivedi, Deepti; Toscano, Sarah; Sharma, Sanjeev; Kolay, Sourav; Raghu, Padinjat

    2017-07-01

    Endocytic turnover is essential for the regulation of the protein composition and function of the plasma membrane, and thus affects the plasma membrane levels of many receptors. In Drosophila melanogaster photoreceptors, photon absorption by the G-protein-coupled receptor (GPCR) rhodopsin 1 (Rh1; also known as NinaE) triggers its endocytosis through clathrin-mediated endocytosis (CME). We find that CME of Rh1 is regulated by phosphatidylinositol 5 phosphate 4-kinase (PIP4K). Flies lacking PIP4K show mislocalization of Rh1 on expanded endomembranes within the cell body. This mislocalization of Rh1 was dependent on the formation of an expanded Rab5-positive compartment. The Rh1-trafficking defect in PIP4K-depleted cells could be suppressed by downregulating Rab5 function or by selectively reconstituting PIP4K in the PI3P-enriched early endosomal compartment of photoreceptors. We also found that loss of PIP4K was associated with increased CME and an enlarged Rab5-positive compartment in cultured Drosophila cells. Collectively, our findings define PIP4K as a novel regulator of early endosomal homeostasis during CME. © 2017. Published by The Company of Biologists Ltd.

  11. Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: A sup 31 P NMR study

    SciTech Connect

    Shashidhar, M.S.; Kuppe, A. ); Volwerk, J.J.; Griffith, O.H.

    1990-09-04

    The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

  12. Effects of adenosine 5'-monophosphate on epidermal turnover.

    PubMed

    Furukawa, Fukumi; Kanehara, Shoko; Harano, Fumiki; Shinohara, Shigeo; Kamimura, Junko; Kawabata, Shigekatsu; Igarashi, Sachiyo; Kawamura, Mitsuaki; Yamamoto, Yuki; Miyachi, Yoshiki

    2008-10-01

    The structure and function of the epidermis is maintained by cell renewal based on epidermal turnover. Epidermal turnover is delayed by aging, and it is thought that the delay of the epidermal turnover is a cause of aging alternation of skin. The epidermal turnover is related to the energy metabolism of epidermal basal cells. Adenosine 5'-triphosphate (ATP) is needed for cell renewal: cell division, and adenosine 5'-monophosphate (AMP) increases the amount of intracellular ATP. These findings suggest that AMP accelerates the epidermal turnover delayed by aging. This study investigated whether AMP and adenosine 5'-monophosphate disodium salt (AMP2Na) accelerates the epidermal turnover. An effect of AMP2Na on cell proliferation was examined by our counting of keratinocytes. An effect of AMP2Na on cell cycle was examined by our counting of basal cells in DNA synthetic period of hairless rats. The effects of AMP2Na (or AMP) on the epidermal turnover were examined by our measuring stratum corneum transit time by use of guinea pigs, and by our measuring stratum corneum surface area by use of hairless rats and in a clinical pharmacological study. The AMP2Na showed two different profiles on the proliferation of primary cultured keratinocytes. At a low concentration it induced cell growth, whereas at a high concentration it inhibited cell growth. The number of basal cells in the DNA synthetic period of AMP2Na was significantly higher than that of the vehicle in hairless rats. The stratum corneum transit time of AMP2Na was significantly shorter than that of the vehicle in guinea pigs. The corneocyte surface area of emulsion containing AMP2Na was significantly smaller than that of the vehicle in volunteers. We conclude that AMP promotes the cell proliferation and the cell cycle progression of epidermal basal cells and accelerates epidermal turnover safely. In addition, AMP is useful for skin rejuvenation in dermatology and aesthetic dermatology.

  13. Intracellular Movement of Green Fluorescent Protein–Tagged Phosphatidylinositol 3-Kinase in Response to Growth Factor Receptor Signaling

    PubMed Central

    Gillham, Helen; Golding, Matthew C.H.M.; Pepperkok, Rainer; Gullick, William J.

    1999-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein–tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein–tagged fragments of p85 we show that binding to the receptor requires the NH2-terminal part of the protein as well as its SH2 domains. PMID:10459020

  14. A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility

    PubMed Central

    Chang, Fanny S.; Han, Gil-Soo; Carman, George M.; Blumer, Kendall J.

    2005-01-01

    Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17ΔWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility. PMID:16216926

  15. A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

    PubMed

    Chang, Fanny S; Han, Gil-Soo; Carman, George M; Blumer, Kendall J

    2005-10-10

    Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

  16. Turnover Among Air Force Nurses.

    DTIC Science & Technology

    1987-03-01

    profession. Journal of Advanced Nursing, 8(3), 227-235. Mowday, R.T. (1984). Strategies for adapting to high rates of employee turnover. Human Resource ...Behavior and Human Performance, 17(l), 66-75. Seybolt, J.W., Pavett, C., & Walker, D.D. (1978). Turnover among nurses: It can be managed . Journal of...committee member, and friend. - John W. Seybolt, Ph.D., Assistant Dean for Graduate Studies, and Professor of Management , School of Business, for his

  17. Differential turnover of phospholipid acyl groups in mouse peritoneal macrophages

    SciTech Connect

    Kuwae, T.; Schmid, P.C.; Johnson, S.B.; Schmid, H.H. )

    1990-03-25

    Phospholipid acyl turnover was assessed in mouse peritoneal exudate cells which consisted primarily of macrophages. The cells were incubated for up to 5 h in media containing 40% H218O, and uptake of 18O into ester carbonyls of phospholipids was determined by gas chromatography-mass spectrometry of hydrogenated methyl esters. The uptake was highest in choline phospholipids and phosphatidylinositol, less in ethanolamine phospholipids, and much less in phosphatidylserine. Acyl groups at the sn-1 and sn-2 positions of diacyl glycerophospholipids, including arachidonic and other long-chain polyunsaturated fatty acids, acquired 18O at about the same rate. Acyl groups of alkylacyl glycerophosphocholine exhibited lower rates of 18O uptake, and acyl groups of ethanolamine plasmalogens (alkenylacyl glycerophosphoethanolamines) acquired only minimal amounts of 18O within 5 h, indicating a low average acyl turnover via free fatty acids. Pulse experiments with exogenous 3H-labeled arachidonic acid supported the concept that acylation of alkenyl glycerophosphoethanolamine occurs by acyl transfer from other phospholipids rather than via free fatty acids and acyl-CoA. The 18O content of intracellular free fatty acids increased gradually over a 5-h period, whereas in extracellular free fatty acids it reached maximal 18O levels within the first hour. Arachidonate and other long-chain polyunsaturated fatty acids were found to participate readily in deacylation-reacylation reactions but were present only in trace amounts in the free fatty acid pools inside and outside the cells. We conclude that acyl turnover of macrophage phospholipids through hydrolysis and reacylation is rapid but tightly controlled so that appreciable concentrations of free arachidonic acid do not occur.

  18. 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

  19. Inositolphosphoglycan mediators structurally related to glycosyl phosphatidylinositol anchors: synthesis, structure and biological activity.

    PubMed

    Martín-Lomas, M; Khiar, N; García, S; Koessler, J L; Nieto, P M; Rademacher, T W

    2000-10-02

    The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators.

  20. Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase.

    PubMed

    Tornieri, Karine; Welshhans, Kristy; Geddis, Matthew S; Rehder, Vincent

    2006-04-01

    Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.

  1. 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.

  2. Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy.

    PubMed

    Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja

    2011-11-11

    Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors.

  3. Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy*

    PubMed Central

    Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja

    2011-01-01

    Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors. PMID:21930714

  4. Isolation of insulin-sensitive phosphatidylinositol-glycan from rat adipocytes. Its impaired breakdown in the streptozotocin-diabetic rat.

    PubMed Central

    Macaulay, S L; Larkins, R G

    1990-01-01

    In this study an insulin-sensitive glycophospholipid from rat adipocytes was isolated and partially characterized. A material that activated pyruvate dehydrogenase was extracted from rat adipocyte membrane supernatants. Its release was stimulated by insulin and phosphatidylinositol-specific-phospholipase C and its activity was destroyed by nitrous acid deamination. These findings suggested that insulin might stimulate breakdown of a glycophospholipid containing inositol and glucosamine, as previously reported for some other cell types [Low & Saltiel (1988) Science 239, 268-275]. A lipid that incorporated [3H]glucosamine, [3H]galactose, [3H]inositol, and [3H]myristate and whose turnover was stimulated by insulin was subsequently isolated from intact adipocytes by sequential t.l.c. using an acidic solvent system followed by a basic solvent system. The effects of insulin on turnover of the lipid in these cells were transient, with maximal effects at 1 min, and there was a typical concentration-response curve to insulin (0.07 nM-7 nM), with effects being detected over the physiological range of insulin concentrations. In contrast with studies in other cells, there was appreciable turnover of the sugar labels. The majority of the [3H]glucosamine and [3H]galactose labels were cycled through to triacylglycerol in the adipocyte. However, of that recovered in the glycophospholipid band, a major proportion (less than 40%) was recovered as the native label. Digestion of the purified molecule with phosphatidylinositol-specific phospholipase C generated a material that activated both pyruvate dehydrogenase and low-Km cyclic AMP phosphodiesterase. Impairment in insulin-stimulated breakdown of the molecule in adipocytes of streptozotocin-diabetic rats was found, consistent with the impaired insulin activation of pyruvate dehydrogenase and glucose utilization seen in this model. These findings suggest that insulin stimulates breakdown of this glycophospholipid by stimulating an

  5. FHL-2 suppresses VEGF-induced phosphatidylinositol 3-kinase/Akt activation via interaction with sphingosine kinase-1.

    PubMed

    Hayashi, Hiroki; Nakagami, Hironori; Takami, Yoichi; Koriyama, Hiroshi; Mori, Masaki; Tamai, Katsuto; Sun, Jianxin; Nagao, Kaori; Morishita, Ryuichi; Kaneda, Yasufumi

    2009-06-01

    In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

  6. Copper-deficient mice have higher cardiac norepinephrine turnover

    SciTech Connect

    Gross, A.M.; Prohaska, J.R. )

    1989-02-01

    Male Swiss albino mice were studied at 6 weeks of age. Their dams were fed a copper-deficient diet (modified AIN-76A) starting 4 days after birth and given deionized water (-Cu) or water with CuSO{sub 4} added (+Cu) (20 {mu}g Cu/ml). When 3 weeks of age mice were weaned and housed in stainless steel cages on the respective treatment of their dams. Turnover of norepinephrine (NE) was studied in 8 experiments using 2 separate techniques. The first procedure used {alpha}-methyl-p-tyrosine methyl ester (300 mg/kg i.p.) to inhibit tyrosine hydroxlase activity. The loss of residual NE was determined by HPLC with electrochemical detection. Regression lines were constructed and fractional turnover (%/h) and calculated turnover (ng/g/h) were determined for heart, cerebellum and adrenal gland. In 4 experiments loss of NE in cerebellum of -Cu ad +Cu mice was equivalent. Loss of NE from adrenal gland could not be detected in the 8 h time course. Loss of NE, both fractional turnover and calculated turnover, from heart of -Cu mice was 4-5 fold higher compared to +Cu controls. A second method using m- hydroxybenzylhydrazine (NSD-1015) (100 mg/kg i.p.), which inhibits aromatic amino acid decarboxylase, confirmed the results. For all 4 experiments the cardiac accumulation of L-DOPA (measured by HPLC) was faster in -Cu mice compared to controls. The higher turnover rate of NE in heart and perhaps other sympathetic nerves may contribute to the higher urinary NE output observed previously.

  7. Resveratrol inhibits polyphosphoinositide metabolism in activated platelets.

    PubMed

    Olas, Beata; Wachowicz, Barbara; Holmsen, Holm; Fukami, Miriam H

    2005-08-15

    The effects of resveratrol (trans-3,4',5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)-P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) were monitored in blood platelets prelabelled with [32P]Pi. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of 32P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)-P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5-P2 available for signalling in these cells.

  8. Phosphatidylinositol-3,4-Bisphosphate and Its Binding Protein Lamellipodin Regulate Chemotaxis of Malignant B Lymphocytes.

    PubMed

    Li, Hongzhao; Wu, Xun; Hou, Sen; Malek, Mouhannad; Kielkowska, Anna; Noh, Edward; Makondo, Kennedy J; Du, Qiujiang; Wilkins, John A; Johnston, James B; Gibson, Spencer B; Lin, Francis; Marshall, Aaron J

    2016-01-15

    Cell migration is controlled by PI3Ks, which generate lipid messengers phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and consequently recruit pleckstrin homology (PH) domain-containing signaling proteins. PI3K inhibition impairs migration of normal and transformed B cells, an effect thought to partly underlie the therapeutic efficacy of PI3K inhibitors in treatment of B cell malignancies such as chronic lymphocytic leukemia. Although a number of studies have implicated phosphatidylinositol-3,4,5-trisphosphate in cell migration, it remains unknown whether PI(3,4)P2 plays a distinct role. Using the PI(3,4)P2-specific phosphatase inositol polyphosphate 4-phosphatase, we investigate the impact of depleting PI(3,4)P2 on migration behavior of malignant B cells. We find that cells expressing wild-type, but not phosphatase dead, inositol polyphosphate 4-phosphatase show impaired SDF-induced PI(3,4)P2 responses and reduced migration in Transwell chamber assays. Moreover, PI(3,4)P2 depletion in primary chronic lymphocytic leukemia cells significantly impaired their migration capacity. PI(3,4)P2 depletion reduced both overall motility and migration directionality in the presence of a stable chemokine gradient. Within chemotaxing B cells, the PI(3,4)P2-binding cytoskeletal regulator lamellipodin (Lpd) was found to colocalize with PI(3,4)P2 on the plasma membrane via its PH domain. Overexpression and knockdown studies indicated that Lpd levels significantly impact migration capacity. Moreover, the ability of Lpd to promote directional migration of B cells in an SDF-1 gradient was dependent on its PI(3,4)P2-binding PH domain. These results demonstrate that PI(3,4)P2 plays a significant role in cell migration via binding to specific cytoskeletal regulators such as Lpd, and they suggest that impairment of PI(3,4)P2-dependent processes may contribute to the therapeutic efficacy of PI3K inhibitors in B cell malignancies.

  9. Phosphatidylinositol turnover (PI) during synaptic activation results from the release of a stimulatory and in inhibitory agonist

    SciTech Connect

    Bencherif, M.; Rubio, R.; Berne, R.M.

    1986-03-05

    PI has been implicated in the process of synaptic transmission and is increased by agonists. It has been suggested that PI is involved in cellular Ca/sup + +/ mobilization and the process represents a series of hydrolytic reactions with inositol as the final product. Hence, the rate of release of /sup 3/H-inositol (/sup 3/H-Ins) from prelabelled inositol phospholipids can be used as an index of PI. In the /sup 3/H-inositol prelabelled frog sympathetic ganglia, they studied the effect of synaptic activity on PI. PI did not change during orthodromic stimulation (20 Hz, 5 min). However, upon cessation of the stimulation, PI increased rapidly and remained elevated for at least 30 min. This increase in PI was reduced by suffusing the ganglia with either acetylcholine or adenosine. In the presence of atropine (5 ..mu..M), orthodromic stimulation increased PI. They hypothesized that synaptic activation releases a long-lasting stimulatory agonist and a short-lived inhibitory (Ach/adenosine) agonist(s) affecting PI. To test this idea, 2 sympathetic ganglia were used. One was prelabelled with /sup 3/H-inositol and the other was not. The two ganglia were placed together in a 5 ..mu..l drop of Ringers solution containing atropine. Orthodromic stimuli were applied to the non-labelled ganglion and elicited release of /sup 3/H-Ins from the non-stimulated ganglion.

  10. Ceramide mediates nanovesicle shedding and cell death in response to phosphatidylinositol ether lipid analogs and perifosine

    PubMed Central

    Gills, J J; Zhang, C; Abu-Asab, M S; Castillo, S S; Marceau, C; LoPiccolo, J; Kozikowski, A P; Tsokos, M; Goldkorn, T; Dennis, P A

    2012-01-01

    Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo. PMID:22764099

  11. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) controls magnesium gatekeeper TRPM6 activity.

    PubMed

    Xie, Jia; Sun, Baonan; Du, Jianyang; Yang, Wenzhong; Chen, Hsiang-Chin; Overton, Jeffrey D; Runnels, Loren W; Yue, Lixia

    2011-01-01

    TRPM6 is crucial for human Mg2+ homeostasis as patients carrying TRPM6 mutations develop hypomagnesemia and secondary hypocalcemia (HSH). However, the activation mechanism of TRPM6 has remained unknown. Here we demonstrate that phosphatidylinositol-4,5-bisphophate (PIP2) controls TRPM6 activation and Mg2+ influx. Stimulation of PLC-coupled M1-receptors to deplete PIP2 potently inactivates TRPM6. Translocation of over-expressed 5-phosphatase to cell membrane to specifically hydrolyze PIP2 also completely inhibits TRPM6. Moreover, depolarization-induced-activation of the voltage-sensitive-phosphatase (Ci-VSP) simultaneously depletes PIP2 and inhibits TRPM6. PLC-activation induced PIP2-depletion not only inhibits TRPM6, but also abolishes TRPM6-mediated Mg2+ influx.Furthermore, neutralization of basic residues in the TRP domain leads to nonfunctional or dysfunctional mutants with reduced activity by PIP2, suggesting that they are likely to participate in interactions with PIP2.Our data indicate that PIP2 is required for TRPM6 channel function; hydrolysis of PIP2 by PLC-coupled hormones/agonists may constitute an important pathway for TRPM6 gating, and perhaps Mg2+ homeostasis.

  12. IPD-196, a novel phosphatidylinositol 3-kinase inhibitor with potent anticancer activity against hepatocellular carcinoma.

    PubMed

    Lee, Ju-Hee; Lee, Hyunseung; Yun, Sun-Mi; Jung, Kyung Hee; Jeong, Yujeong; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

    2013-02-01

    As the activation of phosphatidylinositol 3-kinase (PI3K) is associated with a wide variety of human malignancies, it is emerging as an attractive target for cancer treatment. In this study we synthesized a novel PI3Kα inhibitor, IPD-196 [ethyl 6-(5-(2,4-difluorophenylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate], and evaluated its anticancer effects on human hepatocellular carcinoma (HCC) cells. IPD-196 effectively inhibited the phosphorylation of downstream PI3K effectors such as Akt, mTOR, p70S6K, and 4E-BP1, and its antiproliferative effect was more potent than that of sorafenib or LY294002. It also induced cell cycle arrest at the G0/G1 phase as well as apoptosis by increasing the proportion of sub-G1 apoptotic cells, and the levels of cleaved PARP, caspase-3, and caspase-9. Furthermore, it decreased the expression of HIF-1α and VEGF in Huh-7 cells, and inhibited tube formation and migration of human umbilical vein endothelial cells, which was confirmed by a Matrigel plug assay in mice. Taken together, IPD-196 exhibited its anticancer activity through disruption of the PI3K/Akt pathway that caused cell cycle arrest, apoptosis induction, and inhibition of angiogenesis in human HCC cells. We therefore suggest that IPD-196 may be a potential candidate drug for targeted HCC therapy.

  13. A Phosphatidylinositol 3-Kinase–Pax3 Axis Regulates Brn-2 Expression in Melanoma

    PubMed Central

    Bonvin, Elise; Falletta, Paola; Shaw, Heather; Delmas, Veronique

    2012-01-01

    Deregulation of transcription arising from mutations in key signaling pathways is a hallmark of cancer. In melanoma, the most aggressive and lethal form of skin cancer, the Brn-2 transcription factor (POU3F2) regulates proliferation and invasiveness and lies downstream from mitogen-activated protein kinase (MAPK) and Wnt/β-catenin, two melanoma-associated signaling pathways. In vivo Brn-2 represses expression of the microphthalmia-associated transcription factor, MITF, to drive cells to a more stem cell-like and invasive phenotype. Given the key role of Brn-2 in regulating melanoma biology, understanding the signaling pathways that drive Brn-2 expression is an important issue. Here, we show that inhibition of phosphatidylinositol 3-kinase (PI3K) signaling reduces invasiveness of melanoma cells in culture and strongly inhibits Brn-2 expression. Pax3, a transcription factor regulating melanocyte lineage-specific genes, directly binds and regulates the Brn-2 promoter, and Pax3 expression is also decreased upon PI3K inhibition. Collectively, our results highlight a crucial role for PI3K in regulating Brn-2 and Pax3 expression, reveal a mechanism by which PI3K can regulate invasiveness, and imply that PI3K signaling is a key determinant of melanoma subpopulation diversity. Together with our previous work, the results presented here now place Brn-2 downstream of three melanoma-associated signaling pathways. PMID:22988297

  14. Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels.

    PubMed

    Liu, Bing-Chen; Yang, Li-Li; Lu, Xiao-Yu; Song, Xiang; Li, Xue-Chen; Chen, Guangping; Li, Yichao; Yao, Xincheng; Humphrey, Donald R; Eaton, Douglas C; Shen, Bao-Zhong; Ma, He-Ping

    2015-07-01

    We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K(+) [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCDc14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterol-rich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCDc14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5)P2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I γ [PI(4)P5K I γ], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I γ diffusion into planar regions and elevated PI(4,5)P2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia. Copyright © 2015 by the American Society of Nephrology.

  15. Turnover in the Advancement Profession

    ERIC Educational Resources Information Center

    Iarrobino, Jon D.

    2006-01-01

    Recruitment and retention is an area with which most organizations are concerned. Excessive turnover has exorbitant costs and wastes valuable time. Institutions of higher education are no exception. One of the most vital operations in nonprofit colleges and universities is its Office of Institutional Advancement. More and more, an institution of…

  16. Rac Homologues and Compartmentalized Phosphatidylinositol 4, 5-Bisphosphate Act in a Common Pathway to Regulate Polar Pollen Tube Growth

    PubMed Central

    Kost, Benedikt; Lemichez, Emmanuel; Spielhofer, Pius; Hong, Yan; Tolias, Kimberly; Carpenter, Christopher; Chua, Nam-Hai

    1999-01-01

    Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-δ1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process. PMID:10209027

  17. Measuring Staff Turnover in Nursing Homes

    ERIC Educational Resources Information Center

    Castle, Nicholas G.

    2006-01-01

    Purpose: In this study the levels of staff turnover reported in the nursing home literature (1990-2003) are reviewed, as well as the definitions of turnover used in these prior studies. With the use of primary data collected from 354 facilities, the study addresses the various degrees of bias that result, depending on how staff turnover is defined…

  18. Predictors of Turnover of Female Factory Workers.

    ERIC Educational Resources Information Center

    Koch, James L.; Rhodes, Susan R.

    1981-01-01

    Examines predictors of turnover of female factory workers in a multivariate framework. Findings indicate that organizational, job, and personal characteristics are equally important in explaining turnover. Variables significantly related to turnover are tenure, cycle time, peer leadership, communication flow, training time, family income, and…

  19. How Teacher Turnover Harms Student Achievement

    ERIC Educational Resources Information Center

    Ronfeldt, Matthew; Loeb, Susanna; Wyckoff, James

    2013-01-01

    Researchers and policymakers often assume that teacher turnover harms student achievement, though recent studies suggest this may not be the case. Using a unique identification strategy that employs school-by-grade level turnover and two classes of fixed-effects models, this study estimates the effects of teacher turnover on over 850,000 New York…

  20. A literature review of nursing turnover costs.

    PubMed

    Li, Yin; Jones, Cheryl B

    2013-04-01

    To report the findings of a literature review of studies examining nursing staff turnover costs published between 1990 and 2010. Nurse turnover is a global concern that is both costly for health-care organizations and, in the context of the work environment, affects quality and safety. We reviewed past literature and describe the conceptualization of nurse turnover, evaluate the methodologies and calculation of costs, identify the reported range of turnover costs and provide suggestions for future study. We report inconsistencies in past studies in terms of the conceptualization and measurement of nurse turnover and turnover rates, the methodologies for gathering data and the data sources used, the approaches for calculating turnover costs and the resulting nursing staff turnover costs estimated. Past studies reached different conclusions about nurse turnover. We still need to explore the actual costs and benefits of nurse turnover and retention. This study should be helpful for nurse executives as they build a business case to address nurse turnover in their organizations, and for policy-makers as they develop policies about turnover. © 2012 Blackwell Publishing Ltd.

  1. Using Turnover as a Recruitment Strategy

    ERIC Educational Resources Information Center

    Duncan, Sandra

    2009-01-01

    Teacher turnover is notoriously high in the field of early childhood education with an estimated 33% of staff exiting the workplace each year. Turnover is costly. Not only do high levels of turnover negatively impact children's growth and development, it also erodes the program's economic stability and wherewithal to provide effective operations…

  2. Measuring Staff Turnover in Nursing Homes

    ERIC Educational Resources Information Center

    Castle, Nicholas G.

    2006-01-01

    Purpose: In this study the levels of staff turnover reported in the nursing home literature (1990-2003) are reviewed, as well as the definitions of turnover used in these prior studies. With the use of primary data collected from 354 facilities, the study addresses the various degrees of bias that result, depending on how staff turnover is defined…

  3. Using Turnover as a Recruitment Strategy

    ERIC Educational Resources Information Center

    Duncan, Sandra

    2009-01-01

    Teacher turnover is notoriously high in the field of early childhood education with an estimated 33% of staff exiting the workplace each year. Turnover is costly. Not only do high levels of turnover negatively impact children's growth and development, it also erodes the program's economic stability and wherewithal to provide effective operations…

  4. Estimating Teacher Turnover Costs: A Case Study

    ERIC Educational Resources Information Center

    Levy, Abigail Jurist; Joy, Lois; Ellis, Pamela; Jablonski, Erica; Karelitz, Tzur M.

    2012-01-01

    High teacher turnover in large U.S. cities is a critical issue for schools and districts, and the students they serve; but surprisingly little work has been done to develop methodologies and standards that districts and schools can use to make reliable estimates of turnover costs. Even less is known about how to detect variations in turnover costs…

  5. Phosphatidylinositol Specific Phospholipase C of Plant Stems 1

    PubMed Central

    Pfaffmann, Helmut; Hartmann, Elmar; Brightman, Andrew O.; Morré, D. James

    1987-01-01

    A phosphatidylinositol-specific phospholipase C of plant stems (EC 3.1.4.10) assayed at pH 6.6 and at 30°C cleaved phosphatidylinositol such that more than 85% of the product was inositol-1-phosphate. Other phospholipids were cleaved 5 to 10% or less under these conditions. The phospholipase had both a soluble and a membrane-associated form. The soluble activity accounted for approximately 85 to 90% of the activity and 15% was associated with membranes. The membrane-associated activity was most concentrated in the plasma membranes of hypocotyl segments of both soybean (Glycine max) and bushbean (Phaseolus vulgaris). The plasma membrane location was verified by analysis of highly purified plasma membranes prepared both by aqueous two-phase partitioning and by preparative free-flow electrophoresis and from the quantitation of the activity in all major cell fractions. Internal membranes also contained phospholipase C activity but at specific activity levels of about 0.1 those present in plasma membranes. Golgi apparatus-enriched fractions from which plasma membrane contaminants were removed by two-phase partition contained the activity at specific activity levels 0.2 those of plasma membrane. Both the soluble and the membrane-associated activity was stimulated by calcium but not by calmodulin, either alone or in the presence of calcium. PMID:16665820

  6. Dynamics of Lipid Transfer by Phosphatidylinositol Transfer Proteins in Cells

    PubMed Central

    Shadan, Sadaf; Holic, Roman; Carvou, Nicolas; Ee, Patrick; Li, Michelle; Murray-Rust, Judith; Cockcroft, Shamshad

    2008-01-01

    Of many lipid transfer proteins identified, all have been implicated in essential cellular processes, but the activity of none has been demonstrated in intact cells. Among these, phosphatidylinositol transfer proteins (PITP) are of particular interest as they can bind to and transfer phosphatidylinositol (PtdIns) – the precursor of important signalling molecules, phosphoinositides – and because they have essential functions in neuronal development (PITPα) and cytokinesis (PITPβ). Structural analysis indicates that, in the cytosol, PITPs are in a ‘closed’ conformation completely shielding the lipid within them. But during lipid exchange at the membrane, they must transiently ‘open’. To study PITP dynamics in intact cells, we chemically targeted their C95 residue that, although non-essential for lipid transfer, is buried within the phospholipid-binding cavity, and so, its chemical modification prevents PtdIns binding because of steric hindrance. This treatment resulted in entrapment of open conformation PITPs at the membrane and inactivation of the cytosolic pool of PITPs within few minutes. PITP isoforms were differentially inactivated with the dynamics of PITPβ faster than PITPα. We identify two tryptophan residues essential for membrane docking of PITPs. PMID:18636990

  7. Dynamics of lipid transfer by phosphatidylinositol transfer proteins in cells.

    PubMed

    Shadan, Sadaf; Holic, Roman; Carvou, Nicolas; Ee, Patrick; Li, Michelle; Murray-Rust, Judith; Cockcroft, Shamshad

    2008-09-01

    Of many lipid transfer proteins identified, all have been implicated in essential cellular processes, but the activity of none has been demonstrated in intact cells. Among these, phosphatidylinositol transfer proteins (PITP) are of particular interest as they can bind to and transfer phosphatidylinositol (PtdIns)--the precursor of important signalling molecules, phosphoinositides--and because they have essential functions in neuronal development (PITPalpha) and cytokinesis (PITPbeta). Structural analysis indicates that, in the cytosol, PITPs are in a 'closed' conformation completely shielding the lipid within them. But during lipid exchange at the membrane, they must transiently 'open'. To study PITP dynamics in intact cells, we chemically targeted their C95 residue that, although non-essential for lipid transfer, is buried within the phospholipid-binding cavity, and so, its chemical modification prevents PtdIns binding because of steric hindrance. This treatment resulted in entrapment of open conformation PITPs at the membrane and inactivation of the cytosolic pool of PITPs within few minutes. PITP isoforms were differentially inactivated with the dynamics of PITPbeta faster than PITPalpha. We identify two tryptophan residues essential for membrane docking of PITPs.

  8. Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

    PubMed Central

    Phillips, John H.

    1973-01-01

    Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg2+-dependent and is stimulated by Mn2+ and F− ions. The initial reaction is rapid, with a broad pH profile and a `transition' temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ ions and NaIO4 are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2–3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo. PMID:4360713

  9. Determinants of molecular specificity in phosphoinositide regulation. Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is the endogenous lipid regulating TRPV1.

    PubMed

    Klein, Rebecca M; Ufret-Vincenty, Carmen A; Hua, Li; Gordon, Sharona E

    2008-09-19

    Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P(2)-specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P(3)-specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P(2) inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P(2), and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.

  10. Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling.

    PubMed

    Sato, Masayuki J; Kuwayama, Hidekazu; van Egmond, Wouter N; Takayama, Airi L K; Takagi, Hiroaki; van Haastert, Peter J M; Yanagida, Toshio; Ueda, Masahiro

    2009-04-21

    Switching between attractive and repulsive migration in cell movement in response to extracellular guidance cues has been found in various cell types and is an important cellular function for translocation during cellular and developmental processes. Here we show that the preferential direction of migration during electrotaxis in Dictyostelium cells can be reversed by genetically modulating both guanylyl cyclases (GCases) and the cyclic guanosine monophosphate (cGMP)-binding protein C (GbpC) in combination with the inhibition of phosphatidylinositol-3-OH kinases (PI3Ks). The PI3K-dependent pathway is involved in cathode-directed migration under a direct-current electric field. The catalytic domains of soluble GCase (sGC) and GbpC also mediate cathode-directed signaling via cGMP, whereas the N-terminal domain of sGC mediates anode-directed signaling in conjunction with both the inhibition of PI3Ks and cGMP production. These observations provide an identification of the genes required for directional switching in electrotaxis and suggest that a parallel processing of electric signals, in which multiple-signaling pathways act to bias cell movement toward the cathode or anode, is used to determine the direction of migration.

  11. Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways

    PubMed Central

    Zwang, N. A.; Zhang, R.; Germana, S.; Fan, M. Y.; Hastings, W. D.; Cao, A.; Turka, L. A.

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4+ and CD8+ lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform–specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4+ and CD8+ counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4+ and CD8+ lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity. PMID:27017850

  12. Modulation of high-voltage activated Ca(2+) channels by membrane phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Suh, Byung-Chang; Leal, Karina; Hille, Bertil

    2010-07-29

    Modulation of voltage-gated Ca(2+) channels controls activities of excitable cells. We show that high-voltage activated Ca(2+) channels are regulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) with different sensitivities. Plasma membrane PIP(2) depletion by rapamycin-induced translocation of an inositol lipid 5-phosphatase or by a voltage-sensitive 5-phosphatase (VSP) suppresses Ca(V)1.2 and Ca(V)1.3 channel currents by approximately 35% and Ca(V)2.1 and Ca(V)2.2 currents by 29% and 55%, respectively. Other Ca(V) channels are less sensitive. Inhibition is not relieved by strong depolarizing prepulses. It changes the voltage dependence of channel gating little. Recovery of currents from inhibition needs intracellular hydrolysable ATP, presumably for PIP(2) resynthesis. When PIP(2) is increased by overexpressing PIP 5-kinase, activation and inactivation of Ca(V)2.2 current slow and voltage-dependent gating shifts to slightly higher voltages. Thus, endogenous membrane PIP(2) supports high-voltage activated L-, N-, and P/Q-type Ca(2+) channels, and stimuli that activate phospholipase C deplete PIP(2) and reduce those Ca(2+) channel currents.

  13. Modulation of High-Voltage Activated Ca2+ Channels by Membrane Phosphatidylinositol 4,5-Bisphosphate

    PubMed Central

    Suh, Byung-Chang; Leal, Karina; Hille, Bertil

    2010-01-01

    SUMMARY Modulation of voltage-gated Ca2+ channels controls activities of excitable cells. We show that high-voltage activated Ca2+ channels are regulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP2) with different sensitivities. Plasma membrane PIP2 depletion by rapamycin-induced translocation of an inositol lipid 5-phosphatase or by a voltage-sensitive 5-phosphatase (VSP) suppresses CaV1.2 and CaV1.3 channel currents by ~35%, and CaV2.1 and CaV2.2 currents by 29 and 55%, respectively. Other CaV channels are less sensitive. Inhibition is not relieved by strong depolarizing prepulses. It changes the voltage dependence of channel gating little. Recovery of currents from inhibition needs intracellular hydrolysable ATP, presumably for PIP2 resynthesis. When PIP2 is increased by overexpressing PIP 5-kinase, activation and inactivation of CaV2.2 current slow and voltage-dependent gating shifts to slightly higher voltages. Thus, endogenous membrane PIP2 supports high-voltage activated L-, N-, and P/Q- type Ca2+ channels, and stimuli that activate phospholipase C deplete PIP2 and reduce those Ca2+ channel currents. PMID:20670831

  14. Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling.

    PubMed

    Liu, Kangdong; Park, Chanmi; Chen, Hanyong; Hwang, Joonsung; Thimmegowda, N R; Bae, Eun Young; Lee, Ki Won; Kim, Hong-Gyum; Liu, Haidan; Soung, Nak Kyun; Peng, Cong; Jang, Jae Hyuk; Kim, Kyoon Eon; Ahn, Jong Seog; Bode, Ann M; Dong, Ziming; Kim, Bo Yeon; Dong, Zigang

    2015-09-01

    Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K. © 2014 Wiley Periodicals, Inc.

  15. Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

    PubMed Central

    Liu, Kangdong; Park, Chanmi; Chen, Hanyong; Hwang, Joonsung; Thimmegowda, N.R.; Bae, Eun Young; Lee, Ki Won; Kim, Hong-Gyum; Liu, Haidan; Soung, Nak Kyun; Peng, Cong; Jang, Jae Hyuk; Kim, Kyoon Eon; Ahn, Jong Seog; Bode, Ann M.; Dong, Ziming; Kim, Bo Yeon; Dong, Zigang

    2014-01-01

    Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K. PMID:24700667

  16. EGFR-induced phosphorylation of type Iγ phosphatidylinositol phosphate kinase promotes pancreatic cancer progression

    PubMed Central

    Xue, Junli; Xiong, Xunhao; Huang, Yan; Hu, Jinghua; Ling, Kun

    2017-01-01

    Pancreatic cancer is one of the deadliest malignancies and effective treatment has always been lacking. In current study, we investigated how the type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) participates in the progression of pancreatic ductal adenocarcinoma (PDAC) for novel therapeutic potentials against this lethal disease. We found that PIPKIγ is up-regulated in all tested PDAC cell lines. The growth factor (including EGFR)-induced tyrosine phosphorylation of PIPKIγ is significantly elevated in in situ and metastatic PDAC tissues. Loss of PIPKIγ inhibits the aggressiveness of PDAC cells by restraining the activities of AKT and STAT3, as well as MT1-MMP expression. Therefore when planted into the pancreas of nude mice, PIPKIγ-depleted PDAC cells exhibits substantially repressed tumor growth and metastasis comparing to control PDAC cells. Results from further studies showed that the phosphorylation-deficient PIPKIγ mutant, unlike its wild-type counterpart, cannot rescue PDAC progression inhibited by PIPKIγ depletion. These findings indicate that PIPKIγ, functioning downstream of EGFR signaling, is critical to the progression of PDAC, and suggest that PIPKIγ is potentially a valuable therapeutic target for PDAC treatment. PMID:28388589

  17. Cholesterol modulates ion channels via down-regulation of phosphatidylinositol 4,5-bisphosphate

    PubMed Central

    Chun, Yoon Sun; Shin, Sora; Kim, Yonjung; Cho, Hana; Park, Myoung Kyu; Kim, Tae-Wan; Voronov, Sergey V.; Paolo, Gilbert Di; Suh, Byung-Chang; Chung, Sungkwon

    2010-01-01

    Ubiquitously expressed Mg2+-inhibitory cation (MIC) channels are permeable to Ca2+ and Mg2+ and are essential for cell viability. When membrane cholesterol level was increased by pre-incubating cells with a water-soluble form of cholesterol, the endogenous MIC current in HEK293 cells was negatively regulated. The application of phosphatidylinositol 4,5-bisphosphate (PIP2) recovered MIC current from cholesterol effect. As PIP2 is the direct modulator for MIC channels, high cholesterol content may cause down-regulation of PIP2. To test this possibility, we examined the effect of cholesterol on two exogenously expressed PIP2-sensitive K+ channels: human Ether-a-go-go related gene (HERG) and KCNQ. Enrichment with cholesterol inhibited HERG currents, while inclusion of PIP2 in the pipette solution blocked the cholesterol effect. KCNQ channel was also inhibited by cholesterol. The effects of cholesterol on these channels were blocked by pre-incubating cells with inhibitors for phospholipase C, which may indicate that cholesterol enrichment induces the depletion of PIP2 via phospholipase C activation. Lipid analysis showed that cholesterol enrichment reduced γ-32P incorporation into PIP2 by approximately 35%. Our results suggest that cholesterol may modulate ion channels by changing the levels of PIP2. Thus, an important cross-talk exists among two plasma membrane-enriched lipids, cholesterol and PIP2. PMID:20015154

  18. Turnover: strategies for staff retention.

    PubMed

    SnowAntle, S

    1990-01-01

    This discussion has focused on a number of areas where organizations may find opportunities for more effectively managing employee retention. Given the multitude of causes and consequences, there is no one quick fix. Effective management of employee retention requires assessment of the entire human resources process, that is, recruitment, selection, job design, compensation, supervision, work conditions, etc. Regular and systematic diagnosis of turnover and implementation of multiple strategies and evaluation are needed (Mobley, 1982).

  19. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  20. Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

    PubMed

    Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

  1. Supervisory Turnover in Outpatient Substance Abuse Treatment

    PubMed Central

    Knight, Danica K.; Broome, Kirk M.; Edwards, Jennifer R.; Flynn, Patrick M.

    2009-01-01

    Staff turnover is a significant issue within substance abuse treatment, with implications for service delivery and organizational health. This study examined factors associated with turnover among supervisors in outpatient substance abuse treatment. Turnover was conceptualized as being an individual response to organizational-level influences, and predictors represent aggregate program measures. Participants included 532 staff (including 467 counselors and 65 clinical/program directors) from 90 programs in four regions of the USA. Using logistic regression, analyses of structural factors indicated that programs affiliated with a parent organization and those providing more counseling hours to clients had higher turnover rates. When measures of job attitudes were included, only parent affiliation and collective appraisal of satisfaction were related to turnover. Subsequent analyses identified a trend toward increased supervisory turnover when satisfaction was low following the departure of a previous supervisor. These findings suggest that organizational-level factors can be influential in supervisory turnover. PMID:19949883

  2. Supervisory turnover in outpatient substance abuse treatment.

    PubMed

    Knight, Danica K; Broome, Kirk M; Edwards, Jennifer R; Flynn, Patrick M

    2011-01-01

    Staff turnover is a significant issue within substance abuse treatment, with implications for service delivery and organizational health. This study examined factors associated with turnover among supervisors in outpatient substance abuse treatment. Turnover was conceptualized as being an individual response to organizational-level influences, and predictors represent aggregate program measures. Participants included 532 staff (including 467 counselors and 65 clinical/program directors) from 90 programs in four regions of the USA. Using logistic regression, analyses of structural factors indicated that programs affiliated with a parent organization and those providing more counseling hours to clients had higher turnover rates. When measures of job attitudes were included, only parent affiliation and collective appraisal of satisfaction were related to turnover. Subsequent analyses identified a trend toward increased supervisory turnover when satisfaction was low following the departure of a previous supervisor. These findings suggest that organizational-level factors can be influential in supervisory turnover.

  3. An intrinsic mechanism of secreted protein aging and turnover.

    PubMed

    Yang, Won Ho; Aziz, Peter V; Heithoff, Douglas M; Mahan, Michael J; Smith, Jeffrey W; Marth, Jamey D

    2015-11-03

    The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell-Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease.

  4. An intrinsic mechanism of secreted protein aging and turnover

    PubMed Central

    Yang, Won Ho; Aziz, Peter V.; Heithoff, Douglas M.; Mahan, Michael J.; Smith, Jeffrey W.; Marth, Jamey D.

    2015-01-01

    The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell–Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease. PMID:26489654

  5. Phosphatidylinositol 3- and 4-phosphate are required for normal stomatal movements.

    PubMed

    Jung, Ji-Yul; Kim, Yong-Woo; Kwak, June M; Hwang, Jae-Ung; Young, Jared; Schroeder, Julian I; Hwang, Inhwan; Lee, Youngsook

    2002-10-01

    Phosphatidylinositol (PI) metabolism plays a central role in signaling pathways in both animals and higher plants. Stomatal guard cells have been reported to contain PI 3-phosphate (PI3P) and PI 4-phosphate (PI4P), the products of PI 3-kinase (PI3K) and PI 4-kinase (PI4K) activities. In this study, we tested the roles of PI3P and PI4P in stomatal movements. Both wortmannin (WM) and LY294002 inhibited PI3K and PI4K activities in guard cells and promoted stomatal opening induced by white light or the circadian clock. WM and LY294002 also inhibited stomatal closing induced by abscisic acid (ABA). Furthermore, overexpression in guard cells of GFP:EBD (green fluorescent protein:endosome binding domain of human EEA1) or GFP:FAPP1PH (PI-four-P adaptor protein-1 pleckstrin homology domain), which bind to PI3P and PI4P, respectively, increased stomatal apertures under darkness and white light and partially inhibited stomatal closing induced by ABA. The reduction in ABA-induced stomatal closing with reduced levels of PI monophosphate seemed to be attributable, at least in part, to impaired Ca(2+) signaling, because WM and LY294002 inhibited ABA-induced cytosolic Ca(2+) increases in guard cells. These results suggest that PI3P and PI4P play an important role in the modulation of stomatal closing and that reductions in the levels of functional PI3P and PI4P enhance stomatal opening.

  6. Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol.

    PubMed

    Nickels, J T; Buxeda, R J; Carman, G M

    1994-04-15

    Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles. CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner. Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %). 55-kDa PI 4-kinase activity was not significantly affected by phospholipids. The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells. Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells. However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells. The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells. Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity. The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase. These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol.

  7. Rapid turnover of mitochondrial uncoupling protein 3

    PubMed Central

    Azzu, Vian; Mookerjee, Shona A.; Brand, Martin D.

    2013-01-01

    UCP3 (uncoupling protein 3) and its homologues UCP2 and UCP1 are regulators of mitochondrial function. UCP2 is known to have a short half-life of approx. 1 h, owing to its rapid degradation by the cytosolic 26S proteasome, whereas UCP1 is turned over much more slowly by mitochondrial autophagy. In the present study we investigate whether UCP3 also has a short half-life, and whether the proteasome is involved inUCP3 degradation. UCP3 half-life was examined in the mouse C2C12 myoblast cell line by inhibiting protein synthesis with cycloheximide and monitoring UCP3 protein levels by immunoblot analysis. We show that UCP3 has a short half-life of 0.5–4 h. Rapid degradation was prevented by a cocktail of proteasome inhibitors, supporting a proteasomal mechanism for turnover. In addition, this phenotype is recapitulated in vitro: UCP3 was degraded in mitochondria isolated from rat skeletal muscle or brown adipose tissue with a half-life of 0.5–4 h, but only in the presence of a purified 26S proteasomal fraction. This in vitro proteolysis was also sensitive to proteasome inhibition. This phenotype is in direct contrast with the related proteins UCP1 and the adenine nucleotide translocase, which have long half-lives. Therefore UCP3 is turned over rapidly in multiple cell types in a proteasome-dependent manner. PMID:19954423

  8. Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics

    PubMed Central

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate diverse cellular processes including proliferation, adhesion, survival, and motility. Dysregulated PI3K pathway signaling occurs in one-third of human tumors. Aberrantly activated PI3K signaling also confers sensitivity and resistance to conventional therapies. PI3K has been recognized as an attractive molecular target for novel anti-cancer molecules. In the last few years, several classes of potent and selective small molecule PI3K inhibitors have been developed, and at least fifteen compounds have progressed into clinical trials as new anticancer drugs. Among these, idelalisib has advanced to phase III trials in patients with advanced indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. In this review, we summarized the major molecules of PI3K signaling pathway, and discussed the preclinical models and clinical trials of potent small-molecule PI3K inhibitors. PMID:24261963

  9. Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis

    PubMed Central

    Levin, Roni; Hammond, Gerald R. V.; Balla, Tamas; De Camilli, Pietro; Fairn, Gregory D.; Grinstein, Sergio

    2017-01-01

    We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome. PMID:28035045

  10. 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.

  11. Bone turnover in malnourished children.

    PubMed

    Branca, F; Robins, S P; Ferro-Luzzi, A; Golden, M H

    Pyridinoline (PYD) and deoxypyridinoline (DPD) are cross-linking aminoacids of collagen that are located mainly in bone and cartilage. When bone matrix is resorbed these cross-links are quantitatively excreted in the urine and therefore represent specific markers. We have measured the urinary excretion rate of PYD and DPD in 46 severely malnourished boys to assess their skeletal turnover and to relate this to their subsequent rate of growth. The children were aged 13 months (SD 6), and height-for-age was -3.6 (1.6) Z-score, and weight-for-height was -2.4 (0.8) Z-score. PYD excretion when malnourished and after "recovery" was 11.2 (4.6) nmol h-1m-2 and 32.2 (10.8) nmol h-1m-2 and DPD excretion was 2.6 (1.3) nmol h-1m-2 and 7.5 (3.0) nmol h-1m-2, respectively. The ratio of the two cross-links did not change with recovery. These data show that cartilage and bone turnover is much lower in the malnourished than in the recovered child. There was no difference in the degree of depression of turnover between the children with marasmus, marasmic-kwashiorkor, or kwashiorkor. The rate of height gain during recovery was significantly related to cross-link excretion, age, and weight-for-height on admission. These three factors accounted for 44% of the variance in the height velocity of the children. PYD and DPD excretion rate could be used to assess therapeutic interventions designed to alleviate stunting.

  12. Changes in phosphoinositide turnover, Ca sup 2+ mobilization, and protein phosphorylation in platelets from NIDDM patients

    SciTech Connect

    Ishii, H.; Umeda, F.; Hashimoto, T.; Nawata, H. )

    1990-12-01

    Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration (( Ca2+)i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal (Ca2+)i value was similar in the three groups, the rise in (Ca2+)i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.

  13. Enterococcus faecalis infection activates phosphatidylinositol 3-kinase signaling to block apoptotic cell death in macrophages.

    PubMed

    Zou, Jun; Shankar, Nathan

    2014-12-01

    Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.

  14. Determinants of turnover among nursing department employees.

    PubMed

    Curry, J P; Wakefield, D S; Price, J L; Mueller, C W; McCloskey, J C

    1985-12-01

    A causal model of turnover, or quitting, among hospital nursing department employees was evaluated. This model includes job satisfaction, organizational commitment, and intent to leave as intervening variables that mediate 13 determinants of turnover. The sample consisted of 841 female nursing department employees selected from five hospitals in a western state. Attitudinal and background data were obtained through a mail questionnaire survey, and turnover was monitored for 18 months following the survey. Intent to leave had a strong direct effect on turnover while kinship responsibility, job satisfaction, and organizational commitment had indirect effects on turnover through intent to leave. Task repetitiveness, autonomy, promotional opportunities, and fairness of rewards were important determinants of jobs satisfaction and thus provide a mechanism whereby hospital management may enhance commitment to the organization while reducing turnover.

  15. Enhanced inositide turnover in brain during bicuculline-induced status epilepticus

    SciTech Connect

    Van Rooijen, L.A.; Vadnal, R.; Dobard, P.; Bazan, N.G.

    1986-04-29

    Because brain inositides are enriched in the 1-stearoyl-2-arachidonoyl species, they form a likely source for the tetraenoic free fatty acids (FFA) and diacylglycerols (DG) that are accumulated during seizures. To study inositide turnover during bicuculline-induced seizures, rats were injected intraventricularly and bilaterally with 10-20 microCi /sup 32/P, mechanically ventilated and sacrificed by 6.5 KW head-focused microwave irradiation. Seizure activity was recorded by electroencephalography. Bicuculline-induced seizure activity resulted in: a) almost 50% increase in /sup 32/P labeling of phosphatidic acid (PA); phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2) also increased (24% and 36%, respectively); b) no change in other lipids; and c) water-soluble phosphodiesteratic degradation products, analyzed by high voltage paper electrophoresis, increased 24% in the amount of radiotracer recovered as inositol 1,4-bisphosphate (IP2) and by 44% in the amount recovered as inositol 1,4,5-trisphosphate (IP3). These data indicate that during experimental status epilepticus the cerebral inositide cycle is accelerated: PIP2----(IP3----IP2----IP----I) + DG----PA----PI----PIP----PIP2.

  16. Revisiting nurse turnover costs: adjusting for inflation.

    PubMed

    Jones, Cheryl Bland

    2008-01-01

    Organizational knowledge of nurse turnover costs is important, but gathering these data frequently may not always be feasible in today's fast-paced and complex healthcare environment. The author presents a method to inflation adjust baseline nurse turnover costs using the Consumer Price Index. This approach allows nurse executives to gain current knowledge of organizational nurse turnover costs when primary data collection is not practical and to determine costs and potential savings if nurse retention investments are made.

  17. Intraflagellar transport balances continuous turnover of outer doublet microtubules

    PubMed Central

    Marshall, Wallace F.; Rosenbaum, Joel L.

    2001-01-01

    A central question in cell biology is how cells determine the size of their organelles. Flagellar length control is a convenient system for studying organelle size regulation. Mechanistic models proposed for flagellar length regulation have been constrained by the assumption that flagella are static structures once they are assembled. However, recent work has shown that flagella are dynamic and are constantly turning over. We have determined that this turnover occurs at the flagellar tips, and that the assembly portion of the turnover is mediated by intraflagellar transport (IFT). Blocking IFT inhibits the incorporation of tubulin at the flagellar tips and causes the flagella to resorb. These results lead to a simple steady-state model for flagellar length regulation by which a balance of assembly and disassembly can effectively regulate flagellar length. PMID:11684707

  18. Fat cell turnover in humans.

    PubMed

    Arner, Peter; Spalding, Kirsty L

    2010-05-21

    Obesity is a condition where excess body fat accumulates to such an extent that one's health may be affected. Owing to the cardiovascular and metabolic disorders associated with obesity, and the epidemic of obesity facing most countries today, life expectancy in the developed world may start to decrease for the first time in recent history. Other conditions, such as anorexia nervosa and cachexia, are characterised by subnormal levels of adipose tissue and as with obesity lead to morbidity and mortality. Given the significant personal and economic costs of these conditions and their increasing prevalence in society, understanding the factors that determine the fat mass is therefore of prime interest and may lead to effective treatments and/or interventions for these disorders. Fat mass can be regulated in two ways. The lipid filling of pre-existing fat cells could be altered and the number of fat cells could be changed by the generation of new fat cells or the dying of old ones (i.e. adipocyte turnover). This review summarizes what is known about fat cell turnover in humans and the potential clinical implications. 2010 Elsevier Inc. All rights reserved.

  19. Alcohol, signaling, and ECM turnover.

    PubMed

    Seth, Devanshi; D'Souza El-Guindy, Nympha B; Apte, Minoti; Mari, Montserrat; Dooley, Steven; Neuman, Manuela; Haber, Paul S; Kundu, Gopal C; Darwanto, Agus; de Villiers, Willem J; Vonlaufen, A; Xu, Z; Phillips, P; Yang, S; Goldstein, D; Pirola, R M; Wilson, J S; Moles, Anna; Fernández, Anna; Colell, Anna; García-Ruiz, Carmen; Fernández-Checa, José C; Meyer, Christoph; Meindl-Beinker, Nadja M

    2010-01-01

    Alcohol is recognized as a direct hepatotoxin, but the precise molecular pathways that are important for the initiation and progression of alcohol-induced tissue injury are not completely understood. The current understanding of alcohol toxicity to organs suggests that alcohol initiates injury by generation of oxidative and nonoxidative ethanol metabolites and via translocation of gut-derived endotoxin. These processes lead to cellular injury and stimulation of the inflammatory responses mediated through a variety of molecules. With continuing alcohol abuse, the injury progresses through impairment of tissue regeneration and extracellular matrix (ECM) turnover, leading to fibrogenesis and cirrhosis. Several cell types are involved in this process, the predominant being stellate cells, macrophages, and parenchymal cells. In response to alcohol, growth factors and cytokines activate many signaling cascades that regulate fibrogenesis. This mini-review brings together research focusing on the underlying mechanisms of alcohol-mediated injury in a number of organs. It highlights the various processes and molecules that are likely involved in inflammation, immune modulation, susceptibility to infection, ECM turnover and fibrogenesis in the liver, pancreas, and lung triggered by alcohol abuse.

  20. The costs of turnover in nursing homes.

    PubMed

    Mukamel, Dana B; Spector, William D; Limcangco, Rhona; Wang, Ying; Feng, Zhanlian; Mor, Vincent

    2009-10-01

    Turnover rates in nursing homes have been persistently high for decades, ranging upwards of 100%. To estimate the net costs associated with turnover of direct care staff in nursing homes. DATA AND SAMPLE: Nine hundred two nursing homes in California in 2005. Data included Medicaid cost reports, the Minimum Data Set, Medicare enrollment files, Census, and Area Resource File. We estimated total cost functions, which included in addition to exogenous outputs and wages, the facility turnover rate. Instrumental variable limited information maximum likelihood techniques were used for estimation to deal with the endogeneity of turnover and costs. The cost functions exhibited the expected behavior, with initially increasing and then decreasing returns to scale. The ordinary least square estimate did not show a significant association between costs and turnover. The instrumental variable estimate of turnover costs was negative and significant (P = 0.039). The marginal cost savings associated with a 10% point increase in turnover for an average facility was $167,063 or 2.9% of annual total costs. The net savings associated with turnover offer an explanation for the persistence of this phenomenon over the last decades, despite the many policy initiatives to reduce it. Future policy efforts need to recognize the complex relationship between turnover and costs.

  1. The costs of turnover in nursing homes

    PubMed Central

    Mukamel, Dana B.; Spector, William D.; Limcangco, Rhona; Wang, Ying; Feng, Zhanlian; Mor, Vincent

    2009-01-01

    Background Turnover rates in nursing homes have been persistently high for decades, ranging upwards of 100%. Objectives To estimate the net costs associated with turnover of direct care staff in nursing homes. Data and sample 902 nursing homes in California in 2005. Data included Medicaid cost reports, the Minimum Data Set (MDS), Medicare enrollment files, Census and Area Resource File (ARF). Research Design We estimated total cost functions, which included in addition to exogenous outputs and wages, the facility turnover rate. Instrumental variable (IV) limited information maximum likelihood techniques were used for estimation to deal with the endogeneity of turnover and costs. Results The cost functions exhibited the expected behavior, with initially increasing and then decreasing returns to scale. The ordinary least square estimate did not show a significant association between costs and turnover. The IV estimate of turnover costs was negative and significant (p=0.039). The marginal cost savings associated with a 10 percentage point increase in turnover for an average facility was $167,063 or 2.9% of annual total costs. Conclusion The net savings associated with turnover offer an explanation for the persistence of this phenomenon over the last decades, despite the many policy initiatives to reduce it. Future policy efforts need to recognize the complex relationship between turnover and costs. PMID:19648834

  2. Guide to good practices for operations turnover

    SciTech Connect

    1998-12-01

    This Guide to Good Practices is written to enhance understanding of, and provide direction for, Operations Turnover, Chapter XII of Department of Energy (DOE) Order 5480.19, Conduct of Operations Requirements for DOE Facilities. The practices in this guide should be considered when planning or reviewing operations turnover programs. Contractors are advised to adopt procedures that meet the intent of DOE Order 5480.19. Operations Turnover is an element of an effective Conduct of Operations program. The complexity and array of activities performed in DOE facilities dictate the necessity for a formal operations turnover program to promote safe and efficient operations.

  3. Rapid Turnover of the mTOR Complex 1 (mTORC1) Repressor REDD1 and Activation of mTORC1 Signaling Following Inhibition of Protein Synthesis*

    PubMed Central

    Kimball, Scot R.; Dang Do, An N.; Kutzler, Lydia; Cavener, Douglas R.; Jefferson, Leonard S.

    2009-01-01

    mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription. However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a loss of REDD1 protein due to its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression of mTORC1 activity. PMID:18070882

  4. Phosphatidylinositol 3-kinase mediates the ability of retinol to decrease colorectal cancer cell invasion.

    PubMed

    Lengyel, Jennifer N Griffin; Park, Eun Young; Brunson, Anna R; Pinali, Daniel; Lane, Michelle A

    2014-01-01

    Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor-independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 μM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 μM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P = 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P = 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis.

  5. Phosphatidylinositol phosphate kinase PIPKIγ and phosphatase INPP5E coordinate initiation of ciliogenesis.

    PubMed

    Xu, Qingwen; Zhang, Yuxia; Wei, Qing; Huang, Yan; Hu, Jinghua; Ling, Kun

    2016-02-26

    Defective primary cilia are causative to a wide spectrum of human genetic disorders, termed ciliopathies. Although the regulation of ciliogenesis is intensively studied, how it is initiated remains unclear. Here we show that type Iγ phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (PIPKIγ) and inositol polyphosphate-5-phosphatase E (INPP5E), a Joubert syndrome protein, localize to the centrosome and coordinate the initiation of ciliogenesis. PIPKIγ counteracts INPP5E in regulating tau-tubulin kinase-2 (TTBK2) recruitment to the basal body, which promotes the removal of microtubule capping protein CP110 and the subsequent axoneme elongation. Interestingly, INPP5E and its product--PtdIns(4)P--accumulate at the centrosome/basal body in non-ciliated, but not ciliated, cells. PtdIns(4)P binding to TTBK2 and the distal appendage protein CEP164 compromises the TTBK2-CEP164 interaction and inhibits the recruitment of TTBK2. Our results reveal that PtdIns(4)P homoeostasis, coordinated by PIPKIγ and INPP5E at the centrosome/ciliary base, is vital for ciliogenesis by regulating the CEP164-dependent recruitment of TTBK2.

  6. Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms.

    PubMed

    Kaneko, Kazuma; Ueki, Kohjiro; Takahashi, Noriko; Hashimoto, Shinji; Okamoto, Masayuki; Awazawa, Motoharu; Okazaki, Yukiko; Ohsugi, Mitsuru; Inabe, Kazunori; Umehara, Toshihiro; Yoshida, Masashi; Kakei, Masafumi; Kitamura, Tadahiro; Luo, Ji; Kulkarni, Rohit N; Kahn, C Ronald; Kasai, Haruo; Cantley, Lewis C; Kadowaki, Takashi

    2010-12-01

    Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.

  7. Phosphatidylinositol-specific phospholipase C of Bacillus cereus: cloning, sequencing, and relationship to other phospholipases.

    PubMed Central

    Kuppe, A; Evans, L M; McMillen, D A; Griffith, O H

    1989-01-01

    The phosphatidylinositol (PI)-specific phospholipase C (PLC) of Bacillus cereus was cloned into Escherichia coli by using monoclonal antibody probes raised against the purified protein. The enzyme is specific for hydrolysis of the membrane lipid PI and PI-glycan-containing membrane anchors, which are important structural components of one class of membrane proteins. The protein expressed in E. coli comigrated with B. cereus PI-PLC in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as detected by immunoblotting, and conferred PI-PLC activity on the host. This enzyme activity was inhibited by PI-PLC-specific monoclonal antibodies. The nucleotide sequence of the PI-PLC gene suggests that this secreted bacterial protein is synthesized as a larger precursor with a 31-amino-acid N-terminal extension to the mature enzyme of 298 amino acids. From analysis of coding and flanking sequences of the gene, we conclude that the PI-PLC gene does not reside next to the gene cluster of the other two secreted phospholipases C on the bacterial chromosome. The deduced amino acid sequence of the B. cereus PI-PLC contains a stretch of significant similarity to the glycosylphosphatidylinositol-specific PLC of Trypanosoma brucei. The conserved peptide is proposed to play a role in the function of these enzymes. Images PMID:2509427

  8. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

    PubMed Central

    de Saint-Jean, Maud; Delfosse, Vanessa; Douguet, Dominique; Chicanne, Gaëtan; Payrastre, Bernard; Bourguet, William

    2011-01-01

    Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p–PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers. PMID:22162133

  9. Polycystin-1 Induces Resistance to Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Boca, Manila; Distefano, Gianfranco; Boletta, Alessandra; Qian, Feng; Bhunia, Anil K.; Germino, Gregory G.

    2006-01-01

    Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K β activity in PC-1- expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease. PMID:16452497

  10. ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor identified using the JFCR39 drug discovery system.

    PubMed

    Kong, De-xin; Yamori, Takao

    2010-09-01

    JFCR39 is an informatic anticancer drug discovery system that utilizes a panel of 39 human cancer cells coupled with a drug-activity database. This system not only provides disease-oriented information but can also predict the mechanism of action of a given antitumor agent. Development of a phosphatidylinositol 3-kinase (PI3K) inhibitor as an anticancer drug candidate has attracted a great deal of attention from both academia and industry because PI3K is known to be closely involved in carcinogenesis. ZSTK474 was identified as a PI3K inhibitor using JFCR39 system in combination with COMPARE analysis program. These findings were based on the similar fingerprint (growth inhibition profiles for JFCR39 human cancer cell line panel) with that of a classical PI3K inhibitor LY294002. Biochemical experiments confirmed ZSTK474 to be a potent pan-class I PI3K inhibitor, with high selectivity over other classes of PI3K and protein kinases. We previously reported the in vitro and in vivo antitumor efficacy of ZSTK474, together with the G(0)/G(1) arrest and antiangiogenic activity. Here, we review the JFCR39 system and summarize recent studies on PI3K biology and the development of PI3K inhibitors before discussing ZSTK474 in some detail.

  11. Phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in hyperinsulinemic db/db mice.

    PubMed

    Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

    2012-10-01

    Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.

  12. Selective incorporation of ( sup 15 S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: Agonist-induced deacylation and transformation of stored hydroxyeicosanoids

    SciTech Connect

    Brezinski, M.E.; Serhan, C.N. )

    1990-08-01

    The uptake and mobilization of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a major product of arachidonic acid metabolism, was examined with human neutrophils. Upon exposure to labeled 15-HETE, PMNs rapidly (15 sec to 20 min) incorporated approximately 20% of the label into phosphatidylinositol, while less than 4% was associated with other phospholipid classes and neutral lipids. This pattern was distinct from that of either labeled arachidonate or labeled(5S)-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid (5-HETE), which within 20 min were predominantly associated with triglycerides and phosphatidylcholine. After reversed-phase HPLC, greater than 98% of the label in phosphatidylinositol, isolated from PMNs, was released with phospholipase A2. Upon exposure to either chemotactic peptide (FMLP), phorbol 12-myristate 13-acetate, or an ionophore (A23187), 15-HETE-labeled PMNs released 15-HETE from phosphatidylinositol and displayed an impaired ability to generate leukotriene B4 (LTB4), 20-OH-LTB4, and 20-COOH-LTB4. Deacylated (3H)15-HETE was converted to (5S,15S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid (5,15-DHETE), lipoxin A4, and lipoxin B4, each carrying 3H label. PMNs labeled with 5-HETE also released and transformed this HETE when stimulated. However, the profile of labeled products differed between PMNs with either esterified 15-HETE or 5-HETE. When activated, 5-HETE-labeled PMNs generated both 5,20-DHETE and 5,15-DHETE but not labeled lipoxins. Threshold aggregation induced by FMLP with 15-HETE-labeled PMNs was inhibited, while the threshold response was relatively unimpaired with either A23187 or phorbol 12-myristate 13-acetate-induced aggregation. Results indicate that 15-HETE is esterified into phosphatidylinositol of PMNs, which can be mobilized and transformed upon exposure of the cells to a second signal.

  13. Dynamic Aspects of Voluntary Turnover: An Integrated Approach to Curvilinearity in the Performance-Turnover Relationship

    ERIC Educational Resources Information Center

    Becker, William J.; Cropanzano, Russell

    2011-01-01

    Previous research pertaining to job performance and voluntary turnover has been guided by 2 distinct theoretical perspectives. First, the push-pull model proposes that there is a quadratic or curvilinear relationship existing between these 2 variables. Second, the unfolding model of turnover posits that turnover is a dynamic process and that a…

  14. Dynamic Aspects of Voluntary Turnover: An Integrated Approach to Curvilinearity in the Performance-Turnover Relationship

    ERIC Educational Resources Information Center

    Becker, William J.; Cropanzano, Russell

    2011-01-01

    Previous research pertaining to job performance and voluntary turnover has been guided by 2 distinct theoretical perspectives. First, the push-pull model proposes that there is a quadratic or curvilinear relationship existing between these 2 variables. Second, the unfolding model of turnover posits that turnover is a dynamic process and that a…

  15. Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells.

    PubMed

    Lu, Jing; Caplan, Michael S; Li, Dan; Jilling, Tamas

    2008-05-01

    We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation.

  16. Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells

    PubMed Central

    Lu, Jing; Caplan, Michael S.; Li, Dan; Jilling, Tamas

    2009-01-01

    We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation. PMID:18356536

  17. Molecular Species of Phosphatidylinositol-Cycle Intermediates in the Endoplasmic Reticulum and Plasma Membrane†

    PubMed Central

    Shulga, Yulia V.; Myers, David S.; Ivanova, Pavlina T.; Milne, Stephen B.; Brown, H. Alex; Topham, Matthew K.; Epand, Richard M.

    2009-01-01

    Phosphatidylinositol (PI) turnover is a process requiring both the plasma and ER membranes. We have determined the distribution of phosphatidic acid (PA) and PI and their acyl chain compositions in these two subcellular membranes using mass spectrometry. We assessed the role of PI cycling in determining the molecular species and quantity of these lipids by comparing the compositions of the two membranes isolated from embryonic fibroblasts obtained from diacylglycerol kinase epsilon (DGKε) knock out (KO) and wild type (WT) mice. In the KO cells the conversion of arachidonoyl-rich DAG to PA is blocked by the absence of DGKε, resulting in reducing the rate of PI-cycling. The acyl chain composition is very similar for PI or PA in the endoplasmic reticulum (ER) vs. plasma membrane (PM) and for WT vs. KO. However, the acyl chain profile for PI is very different from that for PA. This indicates that DGKε is not facilitating the direct transfer of a specific species of PA between the PM and the ER. About 20% of the PA in the ER membrane has one short acyl chain of 14 carbons or less. These species of PA are not converted into PI but may play a role in stabilizing regions of high positive curvature in the ER. There are also PI species in both the ER and PM for which there is no detectable PA precursor, indicating that these species of PI are unlikely to arise via the PI-cycle. We find that in the PM of KO cells the levels of PI and of PA are decreased about three-fold in comparison with either the PM of WT cells or in comparison with the ER of KO cells. The PI-cycle is slowed in the KO cells, hence the lipid intermediates of the PI-cycle can no longer be interconverted and are depleted from the PI-cycle by conversion to other species. There is less of an effect of the depletion in the ER where de novo synthesis of PA occurs in comparison with the PM. PMID:20000336

  18. Platelet activation by bacterial phospholipase C involves phosphoinositide turnover and phosphorylation of 47,000 dalton but not 20,000 dalton protein

    SciTech Connect

    Huzoor-Akbar; Anwer, K.

    1986-05-01

    This study was conducted to examine the role of phosphoinositides (PIns) and phosphorylation of 47,000 dalton (P47) and 20,000 dalton (P20) proteins in platelet activation by bacterial phospholipase C (PLC). PLC induced serotonin secretion (SS) and platelet aggregation (PA) in a concentration dependent manner. PLC (0.02 U/ml) caused phosphorylation of P47 in a time dependent manner (27% at 0.5 min to 378% at 7 min). PLC did not induce more than 15% phosphorylation of P20 by 7 min. Aspirin (500 ..mu..M) blocked phosphorylation of P20 but did not inhibit SS, PA or phosphorylation of P47. PLC (0.04 U/ml) decreased radioactivity (cpm) in /sup 32/P labeled phosphatidylinositol (PI), PI-4,5-bis-PO4 (PIP2) and PI-4-PO4 (PIP) by 20%, 12% and 7.5% respectively at 15 sec. The level of PI but not that of PIP2 returned to base line in 3 min. PIP level increased above control values within one min. PLC increased phosphatidic acid level (75% at 0.5 min. to 1545% at 3 min). In other experiments PLC produced diacylglycerol (DAG) in a time and concentration dependent manner. However, no DAG was detectable in the first 60 sec. These data suggest that: (a) PIns turnover and phosphorylation of P47 but not that of P20 is involved in platelet activation by PLC; and (b) DAG production from outer membrane phospholipids is not a prerequisite for platelet activation by PLC.

  19. Modification of erythrocyte membranes by a purified phosphatidylinositol-specific phospholipase C (Staphylococcus aureus).

    PubMed Central

    Low, M G; Finean, J B

    1977-01-01

    A phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was purified by a three-step procedure. The specific activity of the purified enzyme was approx. 6000 times that of the culture supernatant, with an overall recovery of approx. 10%. Estimation of the molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by gel filtration gave values of 33000 and 20000 respectively. A thiol group appears to be necessary for the activity of the enzyme. The purified enzyme had no detectable delta-haemolytic activity and was unable to hydrolyse S. aureus phospholipids. Phosphatidyl-inositol in erythrocyte 'ghosts' was readily hydrolysed by the purified phospholipase C. However, in contrast with our previous preliminary observations, phosphatidylinositol in intact erythrocytes was not significantly hydrolysed. These results suggest that at least 75-80% of the phosphatidylinositol is located at the inner leaflet of the membrane. PMID:849283

  20. Phosphatidylinositol-3 kinase-dependent translational regulation of Id1 involves the PPM1G phosphatase

    PubMed Central

    Xu, Kaiming; Wang, Lanfang; Feng, Wei; Feng, Yue; Shu, Hui-Kuo G.

    2016-01-01

    Id1 is a helix-loop-helix transcriptional modulator that increases the aggressiveness of malignant glial neoplasms. Since most glioblastomas (GBMs) show increased phosphatidylinositol-3 kinase (PI-3K) signaling, we sought to determine whether this pathway regulates Id1 expression. Higher basal Id1 expression correlates with dysregulated PI-3K signaling in multiple established GBM cell lines. Further characterization of PI-3K-dependent Id1 regulation reveals that chemical or genetic inhibition of PI-3K signaling reduces Id1 protein but not mRNA expression. Overall, PI-3K signaling appears to enhance Id1 translation with no significant effect on its stability. PI-3K signaling is known to regulate protein translation through mTORC1-dependent phosphorylation of 4E-BP1, which reduces its association with and inhibition of the translation initiation factor eIF4E. Interestingly, while inhibition of PI-3K and AKT lowers 4E-BP1 phosphorylation and expression of Id1 in all cases, inhibition of TORC1 with rapamycin does not consistently have a similar effect suggesting an alternative mechanism for PI-3K-dependent regulation of Id1 translation. We now identify a potential role for the serine-threonine phosphatase PPM1G in translational regulation of Id1 protein expression. PPM1G knockdown by siRNA increase both 4E-BP1 phosphorylation and Id1 expression and PPM1G and 4E-BP1 co-associates in GBM cells. Furthermore, PPM1G is a phosphoprotein and this phosphorylation appears to be regulated by PI-3K activity. Finally, PI-3K inhibition increases PPM1G activity when assessed by an in vitro phosphatase assay. Our findings provide the first evidence that the PI-3K/AKT signaling pathway modulates PPM1G activity resulting in a shift in the balance between hyper- and hypo-phosphorylated 4E-BP1 and translational regulation of Id1 expression. PMID:27065332

  1. Employee Turnover: An Empirical and Methodological Assessment.

    ERIC Educational Resources Information Center

    Muchinsky, Paul M.; Tuttle, Mark L.

    1979-01-01

    Reviews research on the prediction of employee turnover. Groups predictor variables into five general categories: attitudinal (job satisfaction), biodata, work-related, personal, and test-score predictors. Consistent relationships between common predictor variables and turnover were found for four categories. Eight methodological problems/issues…

  2. Turnover among nursing home staff. A review.

    PubMed

    Cohen-Mansfield, J

    1997-05-01

    Turnover is especially critical in nursing homes: continuity of care and personal relationships between care-givers and residents are important determinants of quality of care. Additionally, for the cognitively impaired nursing home resident, constant change of staff is bound to aggravate disorientation. Research demonstrates links between turnover and employment/employee characteristics and employment availability.

  3. Principal Turnover. Information Capsule. Volume 0914

    ERIC Educational Resources Information Center

    Blazer, Christie

    2010-01-01

    Recent studies indicate that school districts are facing increasing rates of principal turnover. Frequent principal changes deprive schools of the leadership stability they need to succeed, disrupt long-term school reform efforts, and may even be linked to increased teacher turnover and lower levels of student achievement. This Information Capsule…

  4. Employee Turnover: Evidence from a Case Study.

    ERIC Educational Resources Information Center

    Borland, Jeff

    1997-01-01

    Patterns of employee turnover from a medium-sized law firm in Australia were examined in regard to theories of worker mobility (matching, sectoral shift, and incentive). Results support a role for matching effects, but personnel practices affect the timing of turnover. Matching and incentive-based theories do not explain the high rates of turnover…

  5. Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

    PubMed Central

    Higaki, Yasuki; Mikami, Toshio; Fujii, Nobuharu; Hirshman, Michael F.; Koyama, Katsuhiro; Seino, Tetsuya; Tanaka, Keitaro; Goodyear, Laurie J.

    2010-01-01

    We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 μmol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-L-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 μmol/l had no effect on ATP concentrations and did not increase the activities of either the α1 or α2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism. PMID:18303121

  6. Social disadvantage and network turnover.

    PubMed

    Cornwell, Benjamin

    2015-01-01

    Research shows that socially disadvantaged groups--especially African Americans and people of low socioeconomic status (SES)--experience more unstable social environments. I argue that this causes higher rates of turnover within their personal social networks. This is a particularly important issue among disadvantaged older adults, who may benefit from stable networks. This article, therefore, examines whether social disadvantage is related to various aspects of personal network change. Social network change was assessed using longitudinal egocentric network data from the National Social Life, Health, and Aging Project, a study of older adults conducted between 2005 and 2011. Data collection in Wave 2 included a technique for comparing respondents' confidant network rosters between waves. Rates of network losses, deaths, and additions were modeled using multivariate Poisson regression. African Americans and low-SES individuals lost more confidants--especially due to death--than did whites and college-educated respondents. African Americans also added more confidants than whites. However, neither African Americans nor low-SES individuals were able to match confidant losses with new additions to the extent that others did, resulting in higher levels of confidant network shrinkage. These trends are partly, but not entirely, explained by disadvantaged individuals' poorer health and their greater risk of widowhood or marital dissolution. Additional work is needed to shed light on the role played by race- and class-based segregation on group differences in social network turnover. Social gerontologists should examine the role these differences play in explaining the link between social disadvantage and important outcomes in later life, such as health decline. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Social Disadvantage and Network Turnover

    PubMed Central

    2015-01-01

    Objectives. Research shows that socially disadvantaged groups—especially African Americans and people of low socioeconomic status (SES)—experience more unstable social environments. I argue that this causes higher rates of turnover within their personal social networks. This is a particularly important issue among disadvantaged older adults, who may benefit from stable networks. This article, therefore, examines whether social disadvantage is related to various aspects of personal network change. Method. Social network change was assessed using longitudinal egocentric network data from the National Social Life, Health, and Aging Project, a study of older adults conducted between 2005 and 2011. Data collection in Wave 2 included a technique for comparing respondents’ confidant network rosters between waves. Rates of network losses, deaths, and additions were modeled using multivariate Poisson regression. Results. African Americans and low-SES individuals lost more confidants—especially due to death—than did whites and college-educated respondents. African Americans also added more confidants than whites. However, neither African Americans nor low-SES individuals were able to match confidant losses with new additions to the extent that others did, resulting in higher levels of confidant network shrinkage. These trends are partly, but not entirely, explained by disadvantaged individuals’ poorer health and their greater risk of widowhood or marital dissolution. Discussion. Additional work is needed to shed light on the role played by race- and class-based segregation on group differences in social network turnover. Social gerontologists should examine the role these differences play in explaining the link between social disadvantage and important outcomes in later life, such as health decline. PMID:24997286

  8. Clinical development of phosphatidylinositol 3-kinase inhibitors for cancer treatment

    PubMed Central

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer. In recent years, the results of the first phase I clinical trials with PI3K inhibitors have become available. In comparison to other targeted agents such v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors in melanoma or crizotinib in anaplastic lymphoma receptor tyrosine kinase (ALK) translocated tumors, the number of objective responses to PI3K inhibitors is less dramatic. In this review we propose possible strategies to optimize the clinical development of PI3K inhibitors: by exploring the potential role of PI3K isoform-specific inhibitors in improving the therapeutic index, molecular characterization as a basis for patient selection, and the relevance of performing serial tumor biopsies to understand the associated mechanisms of drug resistance. The main focus of this review will be on PI3K isoform-specific inhibitors by describing the functions of different PI3K isoforms, the preclinical activity of selective PI3K isoform-specific inhibitors and the early clinical data of these compounds. PMID:23232172

  9. Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

    PubMed Central

    Baskaran, Sulochanadevi; Carlson, Lars-Anders; Stjepanovic, Goran; Young, Lindsey N; Kim, Do Jin; Grob, Patricia; Stanley, Robin E; Nogales, Eva; Hurley, James H

    2014-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001 PMID:25490155

  10. Glycosyl-phosphatidylinositol molecules of the parasite and the host.

    PubMed

    Ferguson, M A; Brimacombe, J S; Cottaz, S; Field, R A; Güther, L S; Homans, S W; McConville, M J; Mehlert, A; Milne, K G; Ralton, J E

    1994-01-01

    The glycosyl-phosphatidylinositol (GPI) protein-membrane anchors are ubiquitous among the eukaryotes. However, while mammalian cells typically express in the order of 100 thousand copies of GPI-anchor per cell, the parasitic protozoa, particularly the kinetoplastids, express up to 10-20 million copies of GPI-anchor and/or GPI-related glycolipids per cell. Thus GPI-family members dominate the cell surface molecular architecture of these organisms. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of the Leishmania, are known to be essential for parasite survival and infectivity. The highly elevated levels and specialised nature of GPI metabolism in the kinetoplastid parasites suggest that the GPI biosynthetic pathways might be good targets for the development of chemotherapeutic agents. This article introduces the range of GPI structures found in protozoan parasites, and their mammalian hosts, and discusses some aspects of GPI biosynthesis.

  11. Molecular Dynamics Simulations of Phosphatidylinositol Bisphosphate (PIP2)

    NASA Astrophysics Data System (ADS)

    Slochower, David; Janmey, Paul

    2012-02-01

    We are interested in the dynamics of membranes containing the highly charged phospholipid phosphatidylinositol bisphosphate (PIP2 or PtdInsP2). We performed a geometry optimization at the Hartree-Fock 6-31+G* level of theory to determine the biological conformation of the phospholipid headgroup in the presence of water and partial charge distribution. The angle between the headgroup and the acyl chains that form an anchor in the membrane is 94 ^o, indicating that the inositol ring may lie flat along the surface of the inner plasma membrane. Next, we employed hybrid quantum mechanics/molecular mechanics simulations to investigate the protonation state of PIP2 and its interactions with physiological divalent cations such as magnesium and calcium. Based on preliminary data, we propose that the binding of magnesium to PIP2 is mediated by a water molecule that is absent when calcium binds. These results may explain the ability of calcium to induce the formation of PIP2 clusters and phase separation from other phospholipids.

  12. Predictors of turnover intention in nurse faculty.

    PubMed

    Gormley, Denise K; Kennerly, Susan

    2011-04-01

    Turnover of nurse faculty is an increasingly important issue in nursing as the available number of qualified faculty continues to decrease. Understanding the factors that contribute to turnover is important to academic administrators to retain and recruit qualified nursing faculty. The purpose of this study was to examine predictors of turnover intention in nurse faculty working in departments and schools of nursing in Carnegie Doctoral/Research Universities-Extensive, public and private, not-for-profit institutions. The multidimensional model of organizational commitment was used to frame this study. The predictor variables explored were organizational climate, organizational commitment, work role balance, role ambiguity, and role conflict. The work roles examined were research, teaching, and service. Logistical regression was performed to examine the predictors of turnover intention. Organizational climate intimacy and disengagement, affective and continuance organizational commitment, and role ambiguity were shown to predict turnover intention in nurse faculty. Copyright 2011, SLACK Incorporated.

  13. Predictors of physical therapy faculty job turnover.

    PubMed

    Radtka, S

    1993-04-01

    The purpose of this study was to determine what factors are predictive of job turnover of faculty in physical therapy education programs. Four hundred six physical therapy faculty and 92 academic program directors participated in the study. Data were collected from two questionnaires mailed to the participants. Fifteen predictors of turnover were tested, using correlational and multiple regression analyses for data on faculty and education programs. Findings showed that 10% of the faculty resigned within a 1-year period. Low, but significant, correlations were found between higher turnover and fewer years of employment, behavioral intentions to leave, lower salary, higher job stress, and baccalaureate programs. Multiple regression analysis revealed that education programs with faculty having fewer years of employment and the availability of many job alternatives demonstrated significantly higher turnover. Measures to reduce turnover, including faculty recruitment and retention plans, job redesign strategies, and faculty development programs for new faculty, are recommended.

  14. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ

    PubMed Central

    Durand, Nisha; Bastea, Ligia I.; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-01-01

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process. PMID:27775029

  15. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ.

    PubMed

    Durand, Nisha; Bastea, Ligia I; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-10-24

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs' lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process.

  16. A myo-inositol pool utilized for phosphatidylinositol synthesis is depleted in sciatic nerve from rats with streptozotocin-induced diabetes

    SciTech Connect

    Xi Zhu; Eichberg, J. )

    1990-12-01

    Peripheral nerve from experimentally diabetic rats exhibits lowered levels of myo-inositol (MI) and decreased incorporation of ({sup 3}H)MI into phosphatidylinositol (PI). There are indications that diminished PI turnover may be causally related to reduced Na{sup +}, K{sup +} -ATPase activity in diabetic nerve. The authors have investigated whether a metabolic compartment of MI that is essential for PI synthesis is decreased in this tissue. Sciatic nerve segments form streptozotocin-induced diabetic and age-matched normal rats were incubated in vitro with either {sup 32}P{sub i} or ({sup 3}H)cytidine in the presence of propranolol. This cationic amphiphilic agent redirected nerve phospholipid metabolism to produce enhanced {sup 32}P incorporation into PI and decreased labeling of phosphatidylcholine and phosphatidyl-ethanolamine. The incorporation of ({sup 3}H)cytidine into CMP-PA in normal nerve increased up to 15-fold when 0.6 mM propranolol was present. These results strongly suggest the presence in nerve of a pool of MI that is not in equilibrium with the bulk of nerve MI and that is preferentially used for PI synthesis. This metabolic compartment is depleted in diabetic nerve but can be readily replenished by exogenous MI and may correspond to the MI pool that has been proposed to be required for the turnover of a portion of tissue PI involved in maintenance of normal Na{sup +}, K{sup +} -ATPase activity.

  17. MT1-MMP regulates the turnover and endocytosis of extracellular matrix fibronectin.

    PubMed

    Shi, Feng; Sottile, Jane

    2011-12-01

    The extracellular matrix (ECM) is dynamically remodeled by cells during development, normal tissue homeostasis and in a variety of disease processes. We previously showed that fibronectin is an important regulator of ECM remodeling. The deposition and/or polymerization of fibronectin into the ECM controls the deposition and stability of other ECM molecules. In addition, agents that inhibit fibronectin polymerization promote the turnover of fibronectin fibrils and enhance ECM fibronectin endocytosis and intracellular degradation. Endocytosis of ECM fibronectin is regulated by β1 integrins, including α5β1 integrin. We have examined the role of extracellular proteases in regulating ECM fibronectin turnover. Our data show that membrane type matrix metalloproteinase 1 (MT1-MMP; also known as MMP14) is a crucial regulator of fibronectin turnover. Cells lacking MT1-MMP show reduced turnover and endocytosis of ECM fibronectin. MT1-MMP regulates ECM fibronectin remodeling by promoting extracellular cleavage of fibronectin and by regulating α5β1-integrin endocytosis. Our data also show that fibronectin polymerization stabilizes fibronectin fibrils and inhibits ECM fibronectin endocytosis by inhibiting α5β1-integrin endocytosis. These data are the first to show that an ECM protein and its modifying enzyme can regulate integrin endocytosis. These data also show that integrin trafficking plays a major role in modulating ECM fibronectin remodeling. The dual dependence of ECM fibronectin turnover on extracellular proteolysis and endocytosis highlights the complex regulatory mechanisms that control ECM remodeling to ensure maintenance of proper tissue function.

  18. Regulation of endoplasmic reticulum turnover by selective autophagy.

    PubMed

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-06-18

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

  19. Norepinephrine turnover in heart of the copper deficient rat

    SciTech Connect

    Seidel, K.E.; Failla, M.L.; Rosebrough, R. )

    1989-02-01

    Weaned male SD rats were fed a modified AIN-76A diet containing 62% sucrose and either 7 ppm (+Cu) or 0.5 ppm (-Cu) copper for 5 weeks. Dietary copper deprivation resulted in lower concentrations of copper in liver and serum and enlarged hearts. Tissue levels of norepinephrine (NE) and dopamine (DPA) were quantified by HPLC using electrochemical detection. Cardiac concentration of NE and DPA and 26% lower and 63% higher, respectively, in -Cu rats than in +Cu controls. Altered cardiac levels of NE and DPA in -Cu rats were also evident after overnight fasting, a stress that depresses SNS activity. NE turnover was investigated after inhibition of tyrosine hydroxylase by injection of {alpha}-methyl-p-tyrosine methyl ester (250 mg/kg). The fractional rate of NE turnover in the heart was 4.6%/hour for rats fed -Cu and +Cu diets. Calculated NE turnover was greater in heart of +Cu rats than -Cu rats (26 vs. 19 ng/g/hr). NE and DPA concentration in brain, pancreas, and spleen were not affected by dietary copper. These data suggest that synthesis of NE in cardiac nerve endings of the weaned rats sensitive to dietary copper deficiency.

  20. The island-mainland species turnover relationship.

    PubMed

    Stuart, Yoel E; Losos, Jonathan B; Algar, Adam C

    2012-10-07

    Many oceanic islands are notable for their high endemism, suggesting that islands may promote unique assembly processes. However, mainland assemblages sometimes harbour comparable levels of endemism, suggesting that island biotas may not be as unique as is often assumed. Here, we test the uniqueness of island biotic assembly by comparing the rate of species turnover among islands and the mainland, after accounting for distance decay and environmental gradients. We modelled species turnover as a function of geographical and environmental distance for mainland (M-M) communities of Anolis lizards and Terrarana frogs, two clades that have diversified extensively on Caribbean islands and the mainland Neotropics. We compared mainland-island (M-I) and island-island (I-I) species turnover with predictions of the M-M model. If island assembly is not unique, then the M-M model should successfully predict M-I and I-I turnover, given geographical and environmental distance. We found that M-I turnover and, to a lesser extent, I-I turnover were significantly higher than predicted for both clades. Thus, in the first quantitative comparison of mainland-island species turnover, we confirm the long-held but untested assumption that island assemblages accumulate biodiversity differently than their mainland counterparts.

  1. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  2. Vinculin acts as a sensor in lipid regulation of adhesion-site turnover.

    PubMed

    Chandrasekar, Indra; Stradal, Theresia E B; Holt, Mark R; Entschladen, Frank; Jockusch, Brigitte M; Ziegler, Wolfgang H

    2005-04-01

    The dynamics of cell adhesion sites control cell morphology and motility. Adhesion-site turnover is thought to depend on the local availability of the acidic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) can bind to many cell adhesion proteins such as vinculin and talin, but the consequences of this interaction are poorly understood. To study the significance of phospholipid binding to vinculin for adhesion-site turnover and cell motility, we constructed a mutant, vinculin-LD, deficient in acidic phospholipid binding yet with functional actin-binding sites. When expressed in cells, vinculin-LD was readily recruited to adhesion sites, as judged by fluorescence recovery after photobleaching (FRAP) analysis, but cell spreading and migration were strongly impaired, and PIP(2)-dependent disassembly of adhesions was suppressed. Thus, PIP(2) binding is not essential for vinculin activation and recruitment, as previously suggested. Instead, we propose that PIP(2) levels can regulate the uncoupling of adhesion sites from the actin cytoskeleton, with vinculin functioning as a sensor.

  3. Nursing home spending, staffing, and turnover.

    PubMed

    Kash, Bita A; Castle, Nicholas G; Phillips, Charles D

    2007-01-01

    Recent work on nursing home staffing and turnover has stressed the importance of ownership and resources. However, few studies have examined spending behaviors, which might also influence staffing levels and staff turnover rates. This study investigates whether spending behaviors measured by financial ratios are associated with staffing levels and staff turnover in nursing homes. We analyzed cross-sectional data from 1,014 Texas homes. Data were from the 2002 Texas Nursing Facility Medicaid Cost Report and the 2003 Area Resource File. First, we examined differences in financial ratios by ownership type. Next, the effect of 10 financial ratios on staffing levels and turnover rates for registered nurses, licensed vocational nurses, and certified nursing assistants was examined using robust regression models. Descriptive data indicated that expense ratios related to resident care activities and staff development were significantly higher among not-for-profit than for-profit homes. Higher profits were associated with lower staffing levels, but not higher turnover rates. Administrative expenses (a measure of management capacity) had a negative impact both on staffing levels and staff turnover for licensed vocational nurses and certified nursing assistants, but they did not affect registered nurse staffing. Employee benefit expenses exhibited a positive impact on registered nurse and licensed vocational nurse staffing levels. The addition of information on financial ratios to models predicting staffing indicators reduced the effect of ownership on these indicators. Solutions to the staffing and turnover problem should focus on more effective management practices. Certain levels of administrative and staff benefit expenses may be necessary to improve professional staff recruitment and reduce both staffing and turnover costs. Differences in these financial ratios may partially explain the role played by ownership in determining staffing levels and turnover.

  4. Regulation of the PIS1-encoded Phosphatidylinositol Synthase in Saccharomyces cerevisiae by Zinc*

    PubMed Central

    Han, Seung-Hee; Han, Gil-Soo; Iwanyshyn, Wendy M.; Carman, George M.

    2005-01-01

    In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism including an increase in phosphatidylinositol content (Iwanyshyn, W.M., Han, G.-S., and Carman, G.M. (2004) J. Biol. Chem. 279, 21976–21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Δ zrt2Δ mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the β-galactosidase activity driven by the PPIS1-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was due to a transcriptional mechanism. The zinc-mediated induction of the PPIS1-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Δ mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between GST-Zap1p687–880 and a putative UASZRE in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UASZRE in the PIS1 promoter abolished the GST-Zap1p687–880-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene. PMID:15980062

  5. Eps15 homology domain 1-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinositol-(4,5)-bisphosphate and are required for efficient recycling.

    PubMed

    Jović, Marko; Kieken, Fabien; Naslavsky, Naava; Sorgen, Paul L; Caplan, Steve

    2009-06-01

    The C-terminal Eps15 homology domain (EHD) 1/receptor-mediated endocytosis-1 protein regulates recycling of proteins and lipids from the recycling compartment to the plasma membrane. Recent studies have provided insight into the mode by which EHD1-associated tubular membranes are generated and the mechanisms by which EHD1 functions. Despite these advances, the physiological function of these striking EHD1-associated tubular membranes remains unknown. Nuclear magnetic resonance spectroscopy demonstrated that the Eps15 homology (EH) domain of EHD1 binds to phosphoinositides, including phosphatidylinositol-4-phosphate. Herein, we identify phosphatidylinositol-4-phosphate as an essential component of EHD1-associated tubules in vivo. Indeed, an EHD1 EH domain mutant (K483E) that associates exclusively with punctate membranes displayed decreased binding to phosphatidylinositol-4-phosphate and other phosphoinositides. Moreover, we provide evidence that although the tubular membranes to which EHD1 associates may be stabilized and/or enhanced by EHD1 expression, these membranes are, at least in part, pre-existing structures. Finally, to underscore the function of EHD1-containing tubules in vivo, we used a small interfering RNA (siRNA)/rescue assay. On transfection, wild-type, tubule-associated, siRNA-resistant EHD1 rescued transferrin and beta1 integrin recycling defects observed in EHD1-depleted cells, whereas expression of the EHD1 K483E mutant did not. We propose that phosphatidylinositol-4-phosphate is an essential component of EHD1-associated tubules that also contain phosphatidylinositol-(4,5)-bisphosphate and that these structures are required for efficient recycling to the plasma membrane.

  6. Cadherins and Pak1 control contact inhibition of proliferation by Pak1-betaPIX-GIT complex-dependent regulation of cell-matrix signaling.

    PubMed

    Liu, Fengming; Jia, Liwei; Thompson-Baine, Ann-Marie; Puglise, Jason M; Ter Beest, Martin B A; Zegers, Mirjam M P

    2010-04-01

    It is crucial for organ homeostasis that epithelia have effective mechanisms to restrict motility and cell proliferation in order to maintain tissue architecture. On the other hand, epithelial cells need to rapidly and transiently acquire a more mesenchymal phenotype, with high levels of cell motility and proliferation, in order to repair epithelia upon injury. Cross talk between cell-cell and cell-matrix signaling is crucial for regulating these transitions. The Pak1-betaPIX-GIT complex is an effector complex downstream of the small GTPase Rac1. We previously showed that translocation of this complex from cell-matrix to cell-cell adhesion sites was required for the establishment of contact inhibition of proliferation. In this study, we provide evidence that this translocation depends on cadherin function. Cadherins do not recruit the complex by direct interaction. Rather, we found that inhibition of the normal function of cadherin or Pak1 leads to defects in focal adhesion turnover and to increased signaling by phosphatidylinositol 3-kinase. We propose that cadherins are involved in regulation of contact inhibition by controlling the function of the Pak1-betaPIX-GIT complex at focal contacts.

  7. Bone turnover after bariatric surgery.

    PubMed

    Melo, Thalita Lima; Froeder, Leila; Baia, Leandro da Cunha; Heilberg, Ita Pfeferman

    2017-01-01

    The aim of the present study was to evaluate parameters of bone and mineral metabolism after bariatric surgery. This sectional study included data from medical records from 61 bariatric surgery (BS) patients (minimum period of 6 months after the procedure) and from 30 class II and III obese patients as a control group (Cont), consisting of daily dietary intake of macronutrients, calcium and sodium, serum 25(OH)D and parathyroid hormone (PTH) and other biochemical serum and urinary parameters. Bone alkaline phosphatase (BAP), leptin, fibroblast growth factor-23 (FGF-23) and deoxypyridinoline (DPYD) were determined from available banked serum and urinary samples. Mean body mass index (BMI), median energy, carbohydrate, protein and sodium chloride consumption were significantly lower in the BS versus Cont, but calcium and lipids were not. No significant differences were found in ionized calcium, 25(OH)D, PTH and fibroblast growth factor 23 (FGF-23) between groups. Mean serum BAP was significantly higher for BS versus Cont and had a positive correlation with time after the surgical procedure. Mean serum leptin was significantly lower and median urinary DPYD higher in BS versus Cont. The present study showed an increase in bone markers of both bone formation and resorption among bariatric patients up to more than 7 years after the surgical procedure, suggesting that an increased bone turnover persists even at a very long-term follow-up in such patients.

  8. NS5A Inhibitors Impair NS5A–Phosphatidylinositol 4-Kinase IIIα Complex Formation and Cause a Decrease of Phosphatidylinositol 4-Phosphate and Cholesterol Levels in Hepatitis C Virus-Associated Membranes

    PubMed Central

    Reghellin, V.; Donnici, L.; Fenu, S.; Berno, V.; Calabrese, V.; Pagani, M.; Abrignani, S.; Peri, F.

    2014-01-01

    The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex. PMID:25224012

  9. Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

    SciTech Connect

    Baldassare, J.J.; Henderson, P.A.; Fisher, G.J. )

    1989-01-10

    The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulated IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.

  10. The longitudinal study of turnover and the cost of turnover in EMS

    PubMed Central

    Patterson, P. Daniel; Jones, Cheryl B.; Hubble, Michael W.; Carr, Matthew; Weaver, Matthew D.; Engberg, John; Castle, Nicholas

    2010-01-01

    Purpose Few studies have examined employee turnover and associated costs in emergency medical services (EMS). The purpose of this study was to quantify the mean annual rate of turnover, total median cost of turnover, and median cost per termination in a diverse sample of EMS agencies. Methods A convenience sample of 40 EMS agencies was followed over a 6 month period. Internet, telephone, and on-site data collection methods were used to document terminations, new hires, open positions, and costs associated with turnover. The cost associated with turnover was calculated based on a modified version of the Nursing Turnover Cost Calculation Methodology (NTCCM). The NTCCM identified direct and indirect costs through a series of questions that agency administrators answered monthly during the study period. A previously tested measure of turnover to calculate the mean annual rate of turnover was used. All calculations were weighted by the size of the EMS agency roster. The mean annual rate of turnover, total median cost of turnover, and median cost per termination were determined for 3 categories of agency staff mix: all paid staff, mix of paid and volunteer (mixed), and all-volunteer. Results The overall weighted mean annual rate of turnover was 10.7%. This rate varied slightly across agency staffing mix: (all-paid=10.2%, mixed=12.3%, all-volunteer=12.4%). Among agencies that experienced turnover (n=25), the weighted median cost of turnover was $71,613.75, which varied across agency staffing mix: (all-paid=$86,452.05, mixed=$9,766.65, and all-volunteer=$0). The weighted median cost per termination was $6,871.51 and varied across agency staffing mix: (all-paid=$7,161.38, mixed=$1,409.64, and all-volunteer=$0). Conclusions Annual rates of turnover and costs associated with turnover vary widely across types of EMS agencies. The study’s mean annual rate of turnover was lower than expected based on information appearing in the news media and EMS trade magazines. Findings

  11. The longitudinal study of turnover and the cost of turnover in emergency medical services.

    PubMed

    Patterson, P Daniel; Jones, Cheryl B; Hubble, Michael W; Carr, Matthew; Weaver, Matthew D; Engberg, John; Castle, Nicholas

    2010-01-01

    Few studies have examined employee turnover and associated costs in emergency medical services (EMS). To quantify the mean annual rate of turnover, total median cost of turnover, and median cost per termination in a diverse sample of EMS agencies. A convenience sample of 40 EMS agencies was followed over a six-month period. Internet, telephone, and on-site data-collection methods were used to document terminations, new hires, open positions, and costs associated with turnover. The cost associated with turnover was calculated based on a modified version of the Nursing Turnover Cost Calculation Methodology (NTCCM). The NTCCM identified direct and indirect costs through a series of questions that agency administrators answered monthly during the study period. A previously tested measure of turnover to calculate the mean annual rate of turnover was used. All calculations were weighted by the size of the EMS agency roster. The mean annual rate of turnover, total median cost of turnover, and median cost per termination were determined for three categories of agency staff mix: all-paid staff, mix of paid and volunteer (mixed) staff, and all-volunteer staff. The overall weighted mean annual rate of turnover was 10.7%. This rate varied slightly across agency staffing mix (all-paid = 10.2%, mixed = 12.3%, all-volunteer = 12.4%). Among agencies that experienced turnover (n = 25), the weighted median cost of turnover was $71,613.75, which varied across agency staffing mix (all-paid = $86,452.05, mixed = $9,766.65, and all-volunteer = $0). The weighted median cost per termination was $6,871.51 and varied across agency staffing mix (all-paid = $7,161.38, mixed = $1,409.64, and all-volunteer = $0). Annual rates of turnover and costs associated with turnover vary widely across types of EMS agencies. The study's mean annual rate of turnover was lower than expected based on information appearing in the news media and EMS trade magazines. Findings provide estimates of two key

  12. Cytokine Stimulation Promotes Glucose Uptake via Phosphatidylinositol-3 Kinase/Akt Regulation of Glut1 Activity and Trafficking

    PubMed Central

    Wieman, Heather L.; Wofford, Jessica A.

    2007-01-01

    Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization. PMID:17301289

  13. Salt-induced increase in the yield of enzymatically synthesized phosphatidylinositol and the underlying mechanism.

    PubMed

    Muraki, Michiko; Damnjanović, Jasmina; Nakano, Hideo; Iwasaki, Yugo

    2016-09-01

    The purpose of this study was to improve the efficiency of enzymatic synthesis of phosphatidylinositol (PI) from phosphatidylcholine (PC) and myo-inositol in a phospholipase D (PLD)-mediated transphosphatidylation. A conventional biphasic reaction system consisting of ethyl acetate and an aqueous buffer afforded PI with a yield of 14 mol%. In contrast, the reaction performed in the presence of high concentration (0.8-4.3 M) of NaCl in the aqueous phase showed improved PI yield in a NaCl concentration-dependent manner. At 4.3 M NaCl, PI yield of as much as 35 mol% was achieved. The increase in the PI yield offered by other tested salts varied; however, we observed that some salts caused inactivation of the enzyme when used at high concentrations. Although NaCl at high concentration increased the apparent hydrolytic activity on aggregated PC, it decreased the activity towards monomeric PC, indicating that high concentration of salt intrinsically inhibits the enzyme. Binding assays revealed that PLD re-localized from the aqueous phase to the solvent-buffer interface, where the enzymatic reaction takes place, in the presence of both, the salt and PC. Hence, we concluded that improvement of the PI synthesis in the presence of salt occurs mainly due to the accumulation of the enzyme at the interface by strengthening the hydrophobic interactions, by which the apparent activation outweighs the salt-induced inhibitory effect. Using this improved system, several PI with defined structures, namely sn-1, 2-dioleoyl-PI, sn-1-palmitoyl-2-oleoyl-PI, and sn-1-stearoyl-2-arachidonoyl-PI, were successfully synthesized with overall yields of 25-37%, and PI isomeric purities of 91-96%. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Gliotoxin Suppresses Macrophage Immune Function by Subverting Phosphatidylinositol 3,4,5-Trisphosphate Homeostasis

    PubMed Central

    Schlam, Daniel; Canton, Johnathan; Carreño, Marvin; Kopinski, Hannah; Freeman, Spencer A.; Grinstein, Sergio

    2016-01-01

    ABSTRACT Aspergillus fumigatus, an opportunistic fungal pathogen, spreads in the environment by releasing numerous conidia that are capable of reaching the small alveolar airways of mammalian hosts. In otherwise healthy individuals, macrophages are responsible for rapidly phagocytosing and eliminating these conidia, effectively curbing their germination and consequent invasion of pulmonary tissue. However, under some circumstances, the fungus evades phagocyte-mediated immunity and persists in the respiratory tree. Here, we report that A. fumigatus escapes macrophage recognition by strategically targeting phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] metabolism through gliotoxin, a potent immunosuppressive mycotoxin. Time-lapse microscopy revealed that, in response to the toxin, macrophages cease to ruffle, undergo abrupt membrane retraction, and fail to phagocytose large targets effectively. Gliotoxin was found to prevent integrin activation and interfere with actin dynamics, both of which are instrumental for phagocytosis; similar effects were noted in immortalized and primary phagocytes. Detailed studies of the underlying molecular mechanisms of toxicity revealed that inhibition of phagocytosis is attributable to impaired accumulation of PtdIns(3,4,5)P3 and the associated dysregulation of downstream effectors, including Rac and/or Cdc42. Strikingly, in response to the diacylglycerol mimetic phorbol 12-myristate 13-acetate, gliotoxin-treated macrophages reactivate beta integrins, reestablish actin dynamics, and regain phagocytic capacity, despite the overt absence of plasmalemmal PtdIns(3,4,5)P3. Together, our findings identify phosphoinositide metabolism as a critical upstream target of gliotoxin and also indicate that increased diacylglycerol levels can bypass the requirement for PtdIns(3,4,5)P3 signaling during membrane ruffling and phagocytosis. PMID:27048806

  15. Uncoupling by (--)-epigallocatechin-3-gallate of ATP-sensitive potassium channels from phosphatidylinositol polyphosphates and ATP.

    PubMed

    Jin, Jun-Yup; Park, Sung-Hee; Bae, Jae-Hoon; Cho, Ho-Chan; Lim, Jeong-Geun; Park, Won Sun; Han, Jin; Lee, Jin Ho; Song, Dae-Kyu

    2007-09-01

    Of green tea catechins, (--)-epigallocatechin-3-gallate (EGCG) and (--)-epicatechin-3-gallate (ECG), but not (--)-epicatechin and (--)-epigallocatechin, inhibit the activity of ATP-sensitive potassium (K(ATP)) channels at tens of micromolar concentrations, ECG being three times more effective than EGCG. Further, we found that by using cloned beta cell-type K(ATP) channels, only EGCG at 1 microM, a readily achievable plasma concentration by oral intake in humans, but not other epicatechins, significantly blocked channel reactivation after ATP wash-out, suggesting that interaction of phosphatidylinositol polyphosphates (PIP) with the channel was impaired by EGCG. In addition, a 10-fold higher concentration of EGCG reduced the channel sensitivity to ATP, but not AMP and ADP. This effect of EGCG was greater in the channel with the sulfonylurea receptor (SUR) than with the inwardly rectifying K(+) channel (Kir6.2) alone. Neomycin, a polycation, profoundly suppressed the effect of EGCG. Expectedly, glucose-stimulated cytosolic Ca(2+) elevation in rat pancreatic beta cells, and insulin secretory responses to high glucose loading in vivo were impaired by EGCG. In rabbit cardiac myocytes, dinitrophenol-induced opening of the channel was delayed by 1 microM EGCG. These results suggest that EGCG may interact with PIP-binding sites on the Kir6.2 subunit. SUR further endows EGCG with an ability to interfere with an interaction of the gamma-phosphate tail of ATP with Kir6.2. The specificity of EGCG possibly implies that 5'-OH of the B-ring on the pyrogallol moiety in the EGCG molecule may be critical for these actions of EGCG on the K(ATP) channel.

  16. Carbamazepine enhances the activity of glutamate transporter type 3 via phosphatidylinositol 3-kinase.

    PubMed

    Lee, Gwanwoo; Huang, Yueming; Washington, Jacqueline M; Briggs, Nicole W; Zuo, Zhiyi

    2005-01-01

    Glutamate transporters (also called excitatory amino acid transporters, EAAT) participate in maintaining extracellular homeostasis of glutamate, a major excitatory neurotransmitter, and regulating glutamate neurotransmission. EAAT3, the major neuronal EAAT, may also regulate gamma-aminobutyric acid-mediated inhibitory neurotransmission. Dysfunction of EAAT3 has been shown to induce seizure in rats. We hypothesize that carbamazepine, a commonly used antiepileptic agent, enhances EAAT3 activity. We tested this hypothesis using oocytes artificially expressing EAAT3 and C6 rat glioma cells expressing endogenous EAAT3. In oocytes, carbamazepine dose-dependently enhanced EAAT3 activity. The EC50 of this carbamazepine effect was 12.2muM. The concentrations of carbamazepine to significantly enhance EAAT3 activity were within the therapeutic serum levels (17-51muM) of carbamazepine for the antiepileptic effect. Carbamazepine decreased the Km but did not change the maximal response of EAAT3 to glutamate. Carbamazepine-increased EAAT3 activity was inhibited by wortmannin or LY-294002, phosphatidylinositol 3-kinase (PI3K) inhibitors, but was not affected by staurosporine, chelerythrine or calphostin C, protein kinase C inhibitors. In C6 cells, carbamazepine also enhanced the endogenous EAAT3 activity. However, carbamazepine did not affect the activity of EAAT4 expressed in Cos7 cells. These results suggest that carbamazepine at clinically relevant concentrations specifically enhances the affinity of EAAT3 for glutamate to increase EAAT3 activity via a PI3K-dependent pathway. EAAT3 may be a therapeutic target for carbamazepine in the central nervous system.

  17. Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

    PubMed

    Aparicio, I M; Bragado, M J; Gil, M C; Garcia-Herreros, M; Gonzalez-Fernandez, L; Tapia, J A; Garcia-Marin, L J

    2007-08-01

    Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

  18. Supramolecular nanoparticles that target phosphatidylinositol-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy

    PubMed Central

    Kulkarni, Ashish A.; Roy, Bhaskar; Rao, Poornima S.; Wyant, Gregory A.; Mahmoud, Ayaat; Ramachandran, Madhumitha; Sengupta, Poulomi; Goldman, Aaron; Kotamraju, Venkata Ramana; Basu, Sudipta; Mashelkar, Raghunath A; Ruoslahti, Erkki; Dinulescu, Daniela M.; Sengupta, Shiladitya

    2013-01-01

    The centrality of phosphatidylinositol-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intra-tumoral concentration and an insulin resistance ‘class effect’. The current study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG. The supramolecular nanoparticles that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-RasLSL/+/Ptenfl/fl ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the supramolecular nanoparticles highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the supramolecular nanoparticles exerted a temporally-sustained inhibition of phosphorylation of Akt, mTOR, S6K and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of supramolecular nanoparticles abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer treatment

  19. Calcium Directly Regulates Phosphatidylinositol 4,5-Bisphosphate Headgroup Conformation and Recognition

    PubMed Central

    2017-01-01

    The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca2+ is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca2+ is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca2+ with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P2 containing liposomes reveal that Ca2+ as well as Mg2+ reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P2 lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC δ1-PH), is completely inhibited in the presence of Ca2+, while Mg2+ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca2+ binding to the PI(4,5)P2 headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC δ1-PH and PI(4,5)P2, encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms. PMID:28177616

  20. Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Li, Xiaofan; Anishkin, Andriy; Liu, Hansi; van Rossum, Damian B; Chintapalli, Sree V; Sassic, Jessica K; Gallegos, David; Pivaroff-Ward, Kendra; Jegla, Timothy

    2015-11-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K+ channels and voltage-gated Ca2+ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K+ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K+ channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP2 strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP2 has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4-S5 linker) and R479 near the S6 activation gate are required for PIP2 to inhibit voltage activation. The ability of PIP2 to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP2 on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP2-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP2 can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP2 regulation

  1. Coping with Turnovers in School Food Service.

    ERIC Educational Resources Information Center

    Pannell, Dorothy V.

    1988-01-01

    Labor shortages, cost increases, and turnover have prompted Fairfax County Schools, Virginia, food service managers to offer training programs and recruitment bonuses, to use more convenience foods, and to price out every service. (MLF)

  2. Expanding the scope of the turnover flap.

    PubMed

    Mitra, Avir; Spears, Julie; Newsome, Edward; McCampbell, Beth; Kiran, Ravi; Mitra, Amit

    2006-07-01

    Turnover flaps are often utilized as alternatives to more traditional flaps, especially in situations where traditional flap viability is limited. Most turnover flaps are currently used in the lower extremities. This study examined the senior author's use of the turnover flap in 103 cases between 1987 and 2004. Postoperative follow-up ranged from 3 months to 10 years, with an average follow-up of 9 months. The majority (n = 90) of the cases involved the lower extremities and carried high success rates; there were 72 successful operations (complete graft take), 10 partial flap losses (partial graft take that could be treated postoperatively without surgery), and eight complete flap losses (no graft take and the necessity of additional surgery). Three of the partial flap losses and two of the complete flap losses involved patients with end-stage vascular disease. End-stage vascular disease cases represented 20.0 percent of the lower extremity cases and carried a significantly higher percentage of partial or complete flap loss (27.8 percent). These circumstances were examined in detail; the authors found that the turnover flap provided improved outcome to such end-stage patients who otherwise would have undergone amputation. In 13 cases, turnover flaps were utilized in nontraditional regions, such as the chest wall, abdominal wall, head and neck region, and upper extremities, with a high degree of success (zero partial or complete flap losses). These approaches are discussed in detail. The surgical approach is examined with recommendations regarding preferred wound size and type and overall flap design. This study indicates that turnover flaps are effective and useful as an alternative and, in some cases, primary procedure. In addition, the results serve to expand the present scope of the turnover flap by examining nontraditional regions in which the flap was highly successful. The authors believe the turnover flap should be given higher priority as a reconstructive

  3. [Energy turnover of water bugs].

    PubMed

    Waitzbauer, Wolfgang

    1976-06-01

    1. This study concerns the energy turnover of the water bug species Naucoris cimicoides (Naucoridae), Notonecta glauca (Notonectidae) and Ranatra linearis (Nepidae). The results refer to the conditions in the reed belt of the lake "Neusiedler See" in eastern Austria. 2. Population density was, using various methods, quantitatively determined for each test species. In summer the values were as follows: Naucoris 8, Notonecta 2 and Ranatra 0.5 individuals per m(2) in the closed reed belt. Abundance in the next spring was a halving of the initial values due to an increase in the death rate of males in winter. Generally, mortality was very high; the highest death rate for all species occurred in the first two larval stages. The total mortality, beginning at emergence and continuing until immediately after oviposition, was determined to be 91% for Naucoris, 97% for Notonecta and 99% for Ranatra. 3. Production of an average male was 211.45 cal (Naucoris), 243.24 cal (Notonecta) and 256.26 cal (Ranatra) for the entire life span. The production values determined for average females until oviposition are 316.87 cal (Naucoris), 300.79 cal (Notonecta) and 559.51 cal (Ranatra). 53.89 cal (Naucoris), 73.35 cal (Notonecta) and 264.66 cal (Ranatra) are needed for egg production. 4. Respiration was determined by volumetric measurement for all developmental stages and the imago at different times of the year. From emergence until death the following spring the O2-consumption of an average individual was determined as 129.27 cal (♂), 156.45 cal (♀) for Naucoris, 690.66 cal (♂), 882.04 cal (♀) for Notonecta and 548.30 cal (♂), 589.16 cal (♀) for Ranatra. 5. Assimilation was calculated from production and respiration (A=P+R) for all larval and mature stages. Assimilation was determined as 340.72 cal (♂), 419.43 cal (♀) for Naucoris, 933.90 cal (♂), 1109.48 cal (♀) for Notonecta and 804.56 cal (♂), 884.01 cal (♀) for Ranatra, (cumulative values). 6. Since the

  4. Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway.

    PubMed

    Repetto, Emanuela; Yoon, Il-Sang; Zheng, Hui; Kang, David E

    2007-10-26

    Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.

  5. The costs of nurse turnover, part 2: application of the Nursing Turnover Cost Calculation Methodology.

    PubMed

    Jones, Cheryl Bland

    2005-01-01

    This is the second article in a 2-part series focusing on nurse turnover and its costs. Part 1 (December 2004) described nurse turnover costs within the context of human capital theory, and using human resource accounting methods, presented the updated Nursing Turnover Cost Calculation Methodology. Part 2 presents an application of this method in an acute care setting and the estimated costs of nurse turnover that were derived. Administrators and researchers can use these methods and cost information to build a business case for nurse retention.

  6. Amyloid precursor protein controls cholesterol turnover needed for neuronal activity

    PubMed Central

    Pierrot, Nathalie; Tyteca, Donatienne; D'auria, Ludovic; Dewachter, Ilse; Gailly, Philippe; Hendrickx, Aurélie; Tasiaux, Bernadette; Haylani, Laetitia El; Muls, Nathalie; N'Kuli, Francisca; Laquerrière, Annie; Demoulin, Jean-Baptiste; Campion, Dominique; Brion, Jean-Pierre; Courtoy, Pierre J; Kienlen-Campard, Pascal; Octave, Jean-Noël

    2013-01-01

    Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full-length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG-CoA reductase (HMGCR)-mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co-immunoprecipitated and co-localized in the Golgi. This interaction prevented Site-2 protease-mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24-hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity. PMID:23554170

  7. Spatial turnover in the global avifauna

    PubMed Central

    Gaston, Kevin J; Davies, Richard G; Orme, C. David L; Olson, Valerie A; Thomas, Gavin H; Ding, Tzung-Su; Rasmussen, Pamela C; Lennon, Jack J; Bennett, Peter M; Owens, Ian P.F; Blackburn, Tim M

    2007-01-01

    Despite its wide implications for many ecological issues, the global pattern of spatial turnover in the occurrence of species has been little studied, unlike the global pattern of species richness. Here, using a database on the breeding distributions of birds, we present the first global maps of variation in spatial turnover for an entire taxonomic class, a pattern that has to date remained largely a matter of conjecture, based on theoretical expectations and extrapolation of inconsistent patterns from different biogeographic realms. We use these maps to test four predictions from niche theory as to the form that this variation should take, namely that turnover should increase with species richness, towards lower latitudes, and with the steepness of environmental gradients and that variation in turnover is determined principally by rare (restricted) species. Contrary to prediction, we show that turnover is high both in areas of extremely low and high species richness, does not increase strongly towards the tropics, and is related both to average environmental conditions and spatial variation in those conditions. These results are closely associated with a further important and novel finding, namely that global patterns of spatial turnover are driven principally by widespread species rather than the restricted ones. This complements recent demonstrations that spatial patterns of species richness are also driven principally by widespread species, and thus provides an important contribution towards a unified model of how terrestrial biodiversity varies both within and between the Earth's major land masses. PMID:17472910

  8. Spatial turnover in the global avifauna.

    PubMed

    Gaston, Kevin J; Davies, Richard G; Orme, C David L; Olson, Valerie A; Thomas, Gavin H; Ding, Tzung-Su; Rasmussen, Pamela C; Lennon, Jack J; Bennett, Peter M; Owens, Ian P F; Blackburn, Tim M

    2007-07-07

    Despite its wide implications for many ecological issues, the global pattern of spatial turnover in the occurrence of species has been little studied, unlike the global pattern of species richness. Here, using a database on the breeding distributions of birds, we present the first global maps of variation in spatial turnover for an entire taxonomic class, a pattern that has to date remained largely a matter of conjecture, based on theoretical expectations and extrapolation of inconsistent patterns from different biogeographic realms. We use these maps to test four predictions from niche theory as to the form that this variation should take, namely that turnover should increase with species richness, towards lower latitudes, and with the steepness of environmental gradients and that variation in turnover is determined principally by rare (restricted) species. Contrary to prediction, we show that turnover is high both in areas of extremely low and high species richness, does not increase strongly towards the tropics, and is related both to average environmental conditions and spatial variation in those conditions. These results are closely associated with a further important and novel finding, namely that global patterns of spatial turnover are driven principally by widespread species rather than the restricted ones. This complements recent demonstrations that spatial patterns of species richness are also driven principally by widespread species, and thus provides an important contribution towards a unified model of how terrestrial biodiversity varies both within and between the Earth's major land masses.

  9. Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

    PubMed Central

    Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

    2014-01-01

    The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

  10. Modulation of Pathogenic B Cells through Inhibition of Phosphatidylinositol 3-Kinases

    DTIC Science & Technology

    2016-03-01

    of the antibodies bound to the proteins can lodge in the kidneys resulting in damage to the filtering capacity of the kidney . The disease is most...such as nuclear proteins and DNA. These antibodies can cause additional pathologic changes because immune complexes lodge in the kidney which...secreting B cells in a mouse model for lupus, which results in less kidney damage and increased lifespan. 2. KEYWORDS: Lupus, PI3K, B cell, signal

  11. Modulation of Pathogenic B Cells through Inhibition of Phosphatidylinositol 3-Kinases

    DTIC Science & Technology

    2015-10-01

    complexes of the antibodies bound to the proteins can lodge in the kidneys resulting in damage to the filtering capacity of the kidney . The disease...antibodies can cause additional pathologic changes because immune complexes lodge in the kidney which results in further damage. These experiments...lupus, which results in less kidney damage and increased lifespan. 2. KEYWORDS: Provide a brief list of keywords (limit to 20 words). Lupus

  12. Modulation of Pathogenic B Cells through Inhibition of Phosphatidylinositol 3-Kinases

    DTIC Science & Technology

    2014-10-01

    SF298] Note: An abstract is required to be provided in Block 14 This proposal will address the FY12 PRMRP topic area on lupus . Lupus is a life...threatening disease that primarily affects women. Lupus patients develop antibodies that recognize proteins made by the body. This leads to tissue...disease is most often managed using drugs that nonspecifically reduce inflammation and suppress the immune system . However that leaves the patient

  13. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation

    PubMed Central

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  14. 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.

  15. The costs of nurse turnover: part 1: an economic perspective.

    PubMed

    Jones, Cheryl Bland

    2004-12-01

    Nurse turnover is costly for healthcare organizations. Administrators and nurse executives need a reliable estimate of nurse turnover costs and the origins of those costs if they are to develop effective measures of reducing nurse turnover and its costs. However, determining how to best capture and quantify nurse turnover costs can be challenging. Part 1 of this series conceptualizes nurse turnover via human capital theory and presents an update of a previously developed method for determining the costs of nurse turnover, the Nursing Turnover Cost Calculation Method. Part 2 (January 2005) presents a recent application of the methodology in an acute care hospital.

  16. Addressing employee turnover and retention: keeping your valued performers.

    PubMed

    McConnell, Charles R

    2011-01-01

    Employee turnover and employee retention are inextricably linked; to control turnover is to enhance retention. Turnover is a relatively simple concept; however, considerable confusion often results when addressing turnover because of differences in how it is defined; that is, what is counted, how it is counted, and how the turnover rates are expressed. Turnover is also costly, although not enough attention is paid to its cost because so much of it is indirect and thus not readily visible. There are a variety of causes of turnover, some that can be corrected and some that cannot be avoided. Reducing or otherwise controlling turnover requires continuing management attention to its causes and constant recognition of what can and should be controlled and what cannot be controlled. Ongoing attention to turnover is an essential part of the department manager's role; every improvement in turnover is a direct improvement in retention, with eventual positive effects on the bottom line.

  17. Effect of salbutamol on the cerebral levels, uptake and turnover of serotonin.

    PubMed

    Erdö, S L; Kiss, B; Rosdy, B

    1982-03-12

    The effect of salbutamol (SB) on the cerebral levels of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) was determined as well as the effect on the uptake and turnover of 5-HT in rat brain. SB failed to inhibit the cerebral uptake of 5-HT and did not change 5-HT and 5-HIAA levels in the brain; however, it significantly increased the cerebral turnover of 5-HT. The latter effect may partly be responsible for the antidepressant properties of the compounds.

  18. Quantitative Analysis of Actin Turnover in Listeria Comet Tails: Evidence for Catastrophic Filament Turnover

    PubMed Central

    Kueh, Hao Yuan; Brieher, William M.; Mitchison, Timothy J.

    2010-01-01

    Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over. PMID:20923649

  19. Predictors of Staff Turnover and Turnover Intentions within Addiction Treatment Settings: Change Over Time Matters

    PubMed Central

    Garner, Bryan R; Hunter, Brooke D

    2014-01-01

    This study examined the extent to which changes over time in clinicians’ responses to measures of work attitude (eg, job satisfaction) and psychological climate (eg, supervisor support) could predict actual turnover and turnover intentions above and beyond absolute levels of these respective measures. Longitudinal data for this study were collected from a sample of clinicians (N = 96) being trained to implement an evidence-based treatment for adolescent substance use disorders. Supporting findings from a recent staff turnover study, we found job satisfaction change was able to predict actual turnover above and beyond average levels of job satisfaction. Representing new contributions to the staff turnover literature, we also found that change over time in several other key measures (eg, job satisfaction, role manageability, role clarity) explained a significant amount of variance in turnover intentions above and beyond the absolute level of each respective measure. A key implication of the current study is that organizations seeking to improve their ability to assess risk for staff turnover may want to consider assessing staff at multiple points in time in order to identify systematic changes in key employee attitudes like turnover intentions and job satisfaction. PMID:25336960

  20. Regulation of insulin signaling and glucose transporter 4 (GLUT4) exocytosis by phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase, skeletal muscle, and kidney enriched inositol polyphosphate phosphatase (SKIP).

    PubMed

    Ijuin, Takeshi; Takenawa, Tadaomi

    2012-03-02

    The glucose transporter 4 (GLUT4) is responsible for glucose uptake in the skeletal muscle. Insulin-induced translocation of GLUT4 to the plasma membrane requires phosphatidylinositol 3-kinase activation-mediated generation of phosphatidylinositol 3,4,5-trisphosphate PIP(3) and subsequent activation of Akt. Previous studies suggested that skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) has negative effects on the regulation of insulin signaling in the skeletal muscle cells. Here, we compared its effects on insulin signaling by selective inhibition of SKIP, SHIP2, and phosphatase and tensin homologue on chromosome 10 (PTEN) by short interfering RNA in the C2C12 myoblast cells. Suppression of SKIP significantly increased the insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate levels and Akt phosphorylation. Furthermore, silencing of SKIP, but not of PTEN, increased the insulin-dependent recruitment of GLUT4 vesicles to the plasma membrane. Taken together, these results imply that SKIP negatively regulates insulin signaling and glucose uptake by inhibiting GLUT4 docking and/or fusion to the plasma membrane.

  1. Constitutively activated phosphatidylinositol 3-kinase primes platelets from patients with chronic myelogenous leukemia for thrombopoietin-induced aggregation.

    PubMed

    Kubota, Y; Tanaka, T; Ohnishi, H; Kitanaka, A; Okutani, Y; Taminato, T; Ishida, T; Kamano, H

    2004-06-01

    In this study, we examined the effect of thrombopoietin (TPO) on the aggregation of platelets from 40 patients with myeloproliferative disorders (MPDs), including 17 patients with chronic myelogenous leukemia in the chronic phase (CML-CP), 10 with polycythemia vera, 10 with essential thrombocythemia, and three with myelofibrosis. TPO by itself dose-dependently induced the aggregation of platelets from patients with CML-CP but not from those with other MPDs or with CML-CP in cytogenetical complete remission. The expression of CD63 in CML-CP platelets was induced by TPO treatment. Phosphatidylinositol 3-kinase (PI3-kinase) was constitutively activated in CML-CP platelets. Pretreatment with PI3-kinase inhibitors (wortmannin and LY294002) dose-dependently inhibited TPO-induced aggregation of CML-CP platelets. The Abl kinase inhibitor imatinib mesylate and the Jak inhibitor AG490 suppressed TPO-induced aggregation of CML-CP platelets. Pretreatment with imatinib mesylate, but not with AG490, inhibited the activity of PI3-kinase in CML-CP platelets. In addition, tyrosine phosphorylation of Jak2 was undetected in CML-CP platelets before TPO treatment. These findings indicate that the constitutive activation of PI3-kinase primes CML-CP platelets for the aggregation induced by TPO, and that Bcr-Abl, but not Jak family protein tyrosine kinases, are involved in the constitutive activation of PI3-kinase in CML-CP platelets.

  2. Resynthesis of phosphatidylinositol 4,5-bisphosphate mediates adaptation of the caffeine response in rat taste receptor cells.

    PubMed

    Zhao, Fang-Li; Herness, Scott

    2009-01-15

    Caffeine, a prototypic bitter stimulus, produces several physiological actions on taste receptor cells that include inhibition of KIR and KV potassium currents and elevations of intracellular calcium. These responses display adaptation, i.e. their magnitude diminishes in the sustained presence of the stimulus. Levels of the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) are well known to modulate many potassium channels, activating the channel by stabilizing its open state. Here we investigate a putative relationship of KIR and KV with PIP2 levels hypothesizing that inhibition of these currents by caffeine might be allayed by PIP2 resynthesis. Using standard patch-clamp techniques, recordings of either potassium current from rat posterior taste receptor cells produced essentially parallel results when PIP2 levels were manipulated pharmacologically. Increasing PIP2 levels by blocking phosphoinositide-3 kinase with wortmannin or LY294002, or by blocking phospholipase C with U73122 all significantly increased the incidence of adaptation for both KIR and KV. Conversely, lowering PIP2 synthesis by blocking PI4K or using the PIP2 scavengers polylysine or bovine serum albumin reduced the incidence of adaptation. Adaptation could be modulated by activation of protein kinase C but not calcium calmodulin kinase. Collectively, these data support two highly novel conclusions: potassium currents in taste receptor cells are significantly modulated by PIP2 levels and PIP2 resynthesis may play a central role in the gustatory adaptation process at the primary receptor cell level.

  3. Expression signatures of the lipid-based Akt inhibitors phosphatidylinositol ether lipid analogues (PIAs) in NSCLC cells

    PubMed Central

    Zhang, Chunyu; Elkahloun, Abdel G.; Liao, Hongling; Delaney, Shannon; Saber, Barbara; Morrow, Betsy; Gills, Joell J.; Hollander, M. Christine; Prendergast, George C.; Dennis, Phillip A.

    2011-01-01

    Activation of the serine/threonine kinase Akt contributes to the formation, maintenance, and therapeutic resistance of cancer, which is driving development of compounds that inhibit Akt. Phosphatidylinositol ether lipid analogues (PIAs) are analogues of the products of PI3K that inhibit Akt activation, translocation, and the proliferation of a broad spectrum of cancer cell types. To gain insight into the mechanism of PIAs, time-dependent transcriptional profiling of 5 active PIAs and the PI3K inhibitor LY294002 (LY) was performed in NSCLC cells using high-density oligonucleotide arrays. Gene ontology analysis revealed genes involved in apoptosis, wounding response, and angiogenesis were upregulated by PIAs, while genes involved in DNA replication, repair and mitosis were suppressed. Genes that exhibited early differential expression were partitioned into 3 groups; those induced by PIAs only (DUSP1, KLF6, CENTD2, BHLHB2, PREX1), those commonly induced by PIAs and LY (TRIB1, KLF2, RHOB and CDKN1A), and those commonly suppressed by PIAs and LY (IGFBP3, PCNA, PRIM1, MCM3 and HSPA1B). Increased expression of the tumor suppressors RHOB (RhoB), KLF6 (COPEB) and CDKN1A (p21Cip1/Waf1) was validated as an Akt-independent effect that contributed to PIA-induced cytotoxicity. Despite some overlap with LY, active PIAs have a distinct expression signature that contributes to their enhanced cytotoxicity. PMID:21551261

  4. UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity

    PubMed Central

    Sobol, Margarita; Yildirim, Sukriye; Philimonenko, Vlada V; Marášek, Pavel; Castaño, Enrique; Hozák, Pavel

    2013-01-01

    To maintain growth and division, cells require a large-scale production of rRNAs which occurs in the nucleolus. Recently, we have shown the interaction of nucleolar phosphatidylinositol 4,5-bisphosphate (PIP2) with proteins involved in rRNA transcription and processing, namely RNA polymerase I (Pol I), UBF, and fibrillarin. Here we extend the study by investigating transcription-related localization of PIP2 in regards to transcription and processing complexes of Pol I. To achieve this, we used either physiological inhibition of transcription during mitosis or inhibition by treatment the cells with actinomycin D (AMD) or 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB). We show that PIP2 is associated with Pol I subunits and UBF in a transcription-independent manner. On the other hand, PIP2/fibrillarin colocalization is dependent on the production of rRNA. These results indicate that PIP2 is required not only during rRNA production and biogenesis, as we have shown before, but also plays a structural role as an anchor for the Pol I pre-initiation complex during the cell cycle. We suggest that throughout mitosis, PIP2 together with UBF is involved in forming and maintaining the core platform of the rDNA helix structure. Thus we introduce PIP2 as a novel component of the NOR complex, which is further engaged in the renewed rRNA synthesis upon exit from mitosis. PMID:24513678

  5. Artesunate Protected Blood-Brain Barrier via Sphingosine 1 Phosphate Receptor 1/Phosphatidylinositol 3 Kinase Pathway After Subarachnoid Hemorrhage in Rats.

    PubMed

    Zuo, Shilun; Ge, Hongfei; Li, Qiang; Zhang, Xuan; Hu, Rong; Hu, Shengli; Liu, Xin; Zhang, John H; Chen, Yujie; Feng, Hua

    2017-03-01

    Blood-brain barrier preservation plays an important role in attenuating vasogenic brain edema after subarachnoid hemorrhage (SAH). This study was designed to investigate the protective effect and mechanism of artesunate, a traditional anti-malaria drug, on blood-brain barrier after SAH. Three hundred and seventy-seven (377) male Sprague-Dawley rats were subjected to endovascular perforation model for SAH. The rats received artesunate alone or in combination with Sphingosine-1-phosphate receptor-1 (S1P1) small interfering RNA (siRNA), antagonist VPC23019, or phosphatidylinositol 3-kinase inhibitor wortmannin after SAH. Modified Garcia score, SAH grades, brain water content, Evans blue leakage, transmission electron microscope, immunohistochemistry staining, Western blot, and cultured endothelial cells were used to investigate the optimum concentration and the therapeutic mechanism of artesunate. We found that artesunate (200 mg/kg) could do better in raising modified Garcia score, reducing brain water content and Evans blue leakage than other groups after SAH. Moreover, artesunate elevated S1P1 expression, enhanced phosphatidylinositol 3-kinase activation, lowered GSK-3β activation, stabilized β-catenin, and improved the expression of Claudin-3 and Claudin-5 after SAH in rats. These effects were eliminated by S1P1 siRNA, VPC23019, and wortmannin. This study revealed that artesunate could preserve blood-brain barrier integrity and improve neurological outcome after SAH, possibly through activating S1P1, enhancing phosphatidylinositol 3-kinase activation, stabilizing β-catenin via GSK-3β inhibition, and then effectively raising the expression of Claudin-3 and Claudin-5. Therefore, artesunate may be favorable for the blood-brain barrier (BBB) protection after SAH and become a potential candidate for the treatment of SAH patients.

  6. Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors.

    PubMed

    Alswied, Abdullah; Parekh, Anant B

    2015-12-04

    Oscillations in cytoplasmic Ca(2+) concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca(2+) oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca(2+)-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca(2+) oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li(+), an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li(+)-treated cells, cytoplasmic Ca(2+) signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca(2+) oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca(2+) oscillations, and these could not be rescued by inositol or PI4P. In Li(+)-treated cells, recovery of the cytoplasmic Ca(2+) oscillations in the presence of inositol or PI4P was suppressed when Ca(2+) influx through store-operated Ca(2+) channels was inhibited. After rundown of the Ca(2+) signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca(2+) release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca(2+) entry is needed to sustain cytoplasmic Ca(2+) signaling following leukotriene receptor activation both by refilling the Ca(2+) stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.

  7. Nurse turnover: the mediating role of burnout.

    PubMed

    Leiter, Michael P; Maslach, Christina

    2009-04-01

    This study tested whether the mediation model of burnout could predict nurses' turnover intentions. A better understanding of what factors support a commitment to a nursing career could inform both policies and workplace practices. The mediation model of burnout provides a way of linking the quality of a nurse's worklife to various outcomes, such as turnover. Data on areas of worklife, burnout, and turnover intentions were collected by surveying 667 Canadian nurses in the Atlantic Provinces. The findings supported the mediation model of burnout, in which areas of worklife predicted burnout, which in turn predicted turnover intentions. Cynicism was the key burnout dimension for turnover, and the most critical areas of worklife were value conflicts and inadequate rewards. The results of this study provide some new insights into how the intention of nurses to leave their job is related to particular aspects of their worklife and to burnout. These results suggest what may be the most appropriate areas to target for interventions to reduce the risk of nurses exiting early from their chosen career.

  8. Primary care physician job satisfaction and turnover.

    PubMed

    Buchbinder, S B; Wilson, M; Melick, C F; Powe, N R

    2001-07-01

    To examine the relationship of personal characteristics, organizational characteristics, and overall job satisfaction to primary care physician (PCP) turnover. A cohort of 507 postresident, nonfederally employed PCPs younger than 45 years of age, who completed their medical training between 1982 and 1985, participated in national surveys in 1987 and 1991. Psychological, economic, and sociological theories and constructs provided a conceptual framework. Primary care physician personal, organizational, and overall job satisfaction variables from 1987 were considered independent variables. Turnover-related responses from 1991 were dependent variables. Bivariate and multivariate analyses were conducted. More than half (55%) of all PCPs in the cohort left at least 1 practice between 1987 and 1991. Twenty percent of the cohort left 2 employers. PCPs dissatisfied in 1987 were 2.38 times more likely to leave (P < .001). Primary care physicians who believed that third-party payer influence would decrease in 5 years were 1.29 times more likely to leave (P < .03). Non-board certified PCPs were 1.3 times more likely to leave (P < .003). Primary care physicians who believed that standardized protocols were overused were 1.18 times more likely to leave (P < .05). Specialty, gender, age, race, and practice setting were not associated with PCP turnover. Turnover was an important phenomenon among PCPs in this cohort. The results of this study could enable policy makers, managed care organizations, researchers, and others to better understand the relationship between job satisfaction and turnover.

  9. Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.

    PubMed

    Chandrasekar, Bysani; Melby, Peter C; Sarau, Henry M; Raveendran, Muthuswamy; Perla, Rao P; Marelli-Berg, Federica M; Dulin, Nickolai O; Singh, Ishwar S

    2003-02-14

    It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.

  10. Nursing home staffing, turnover, and case mix.

    PubMed

    Harrington, Charlene; Swan, James H

    2003-09-01

    This study examined the predictors of total nurse and registered nurse (RN) staffing hours per resident day separately in all free-standing California nursing homes (1,555), using staffing data from state cost reports in 1999. This study used a two-stage least squares model, taking into account nursing turnover rates, resident case mix levels, and other factors. As expected, total nurse and RN staffing hours were negatively associated with nurse staff turnover rates and positively associated with resident case mix. Facilities were resource dependent in that a high proportion of Medicare residents predicted higher staffing hours, and a higher proportion of Medicaid residents predicted lower staffing hours and higher turnover rates. Nursing assistant wages were positively associated with total nurse staffing hours. For-profit facilities and high-occupancy rate facilities had lower total nurse and RN staffing hours. Medicaid reimbursement rates and multifacility organizations were positively associated with RN staffing hours.

  11. Changes of Protein Turnover in Aging Caenorhabditis elegans.

    PubMed

    Dhondt, Ineke; Petyuk, Vladislav A; Bauer, Sophie; Brewer, Heather M; Smith, Richard D; Depuydt, Geert; Braeckman, Bart P

    2017-09-01

    Protein turnover rates severely decline in aging organisms, including C. elegans However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse (15)N-labeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, whereas only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, whereas randomly diverging turnover patterns with age were the norm. Our data suggests increased heterogeneity of protein turnover of the translation machinery, whereas protein turnover of ubiquitin-proteasome and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Type III phosphatidylinositol 4 kinases: structure, function, regulation, signalling and involvement in disease.

    PubMed

    Dornan, Gillian L; McPhail, Jacob A; Burke, John E

    2016-02-01

    Many important cellular functions are regulated by the selective recruitment of proteins to intracellular membranes mediated by specific interactions with lipid phosphoinositides. The enzymes that generate lipid phosphoinositides therefore must be properly positioned and regulated at their correct cellular locations. Phosphatidylinositol 4 kinases (PI4Ks) are key lipid signalling enzymes, and they generate the lipid species phosphatidylinositol 4-phosphate (PI4P), which plays important roles in regulating physiological processes including membrane trafficking, cytokinesis and organelle identity. PI4P also acts as the substrate for the generation of the signalling phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI4Ks also play critical roles in a number of pathological processes including mediating replication of a number of pathogenic RNA viruses, and in the development of the parasite responsible for malaria. Key to the regulation of PI4Ks is their regulation by a variety of both host and viral protein-binding partners. We review herein our current understanding of the structure, regulatory interactions and role in disease of the type III PI4Ks.

  13. Drug-resistant phosphatidylinositol 3-kinase: guidance for the preemptive strike.

    PubMed

    Vogt, Peter K

    2008-08-12

    In this issue of Cancer Cell, Zunder et al. (2008) describe surprising findings from investigating inhibitor-resistant mutations in the affinity pocket of p110 alpha of phosphatidylinositol 3-kinase (PI3K). Information on these critical residues provides a road map for generating novel PI3K inhibitors that can overcome the anticipated resistance mutations.

  14. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.

    PubMed

    Lacalle, Rosa Ana; de Karam, Juan C; Martínez-Muñoz, Laura; Artetxe, Ibai; Peregil, Rosa M; Sot, Jesús; Rojas, Ana M; Goñi, Félix M; Mellado, Mario; Mañes, Santos

    2015-06-01

    Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity.

  15. Regulation of the PIS1-encoded phosphatidylinositol synthase in Saccharomyces cerevisiae by zinc.

    PubMed

    Han, Seung-Hee; Han, Gil-Soo; Iwanyshyn, Wendy M; Carman, George M

    2005-08-12

    In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976-21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Delta zrt2Delta mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the beta-galactosidase activity driven by the P(PIS1)-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the P(PIS1)-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Delta mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p(687-880) and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p(687-880)-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.

  16. Clinical development of phosphatidylinositol-3 kinase pathway inhibitors.

    PubMed

    Arteaga, Carlos L

    2010-01-01

    The PI3K pathway is the most commonly altered in human cancer. Several recent phase I studies with therapeutic inhibitors of this pathway have shown that pharmacological inhibition of PI3K in humans is feasible and overall well tolerated. Furthermore, there has already been clinical evidence of anti-tumor activity in patients with advanced cancer. The intensity and duration of PI3K inhibition required for an antitumor effect and the optimal pharmacodynamic biomarker(s) of pathway inactivation remain to be established. Preclinical and early clinical data support focusing on trials with PI3K inhibitors that are at a minimum enriched with patients with alterations in this signaling pathway. These inhibitors are likely to be more effective in combination with established and other novel molecular therapies.

  17. Regulation of Insulin Secretion by Phosphatidylinositol-4,5-Bisphosphate

    PubMed Central

    Tomas, Alejandra; Yermen, Barbara; Regazzi, Romano; Pessin, Jeffrey E.; Halban, Philippe A.

    2014-01-01

    The role of PIP2 in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP2 with PH-PLC-GFP or PIP5KIγ RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIγ improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP2, transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP2 co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIγ-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIγ, while blocking PIP2 reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP2 plays a pro-survival role in MIN6B1 cells, excessive PIP2 production due to PIP5KIγ over-expression inhibits secretion due to both a defective Arf6/PIP5KIγ-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIγ-dependent perturbation of F-actin cytoskeleton remodeling. PMID:19845918

  18. Quantification of isotopic turnover in agricultural systems

    NASA Astrophysics Data System (ADS)

    Braun, A.; Auerswald, K.; Schnyder, H.

    2012-04-01

    The isotopic turnover, which is a proxy for the metabolic rate, is gaining scientific importance. It is quantified for an increasing range of organisms, from microorganisms over plants to animals including agricultural livestock. Additionally, the isotopic turnover is analyzed on different scales, from organs to organisms to ecosystems and even to the biosphere. In particular, the quantification of the isotopic turnover of specific tissues within the same organism, e.g. organs like liver and muscle and products like milk and faeces, has brought new insights to improve understanding of nutrient cycles and fluxes, respectively. Thus, the knowledge of isotopic turnover is important in many areas, including physiology, e.g. milk synthesis, ecology, e.g. soil retention time of water, and medical science, e.g. cancer diagnosis. So far, the isotopic turnover is quantified by applying time, cost and expertise intensive tracer experiments. Usually, this comprises two isotopic equilibration periods. A first equilibration period with a constant isotopic input signal is followed by a second equilibration period with a distinct constant isotopic input signal. This yields a smooth signal change from the first to the second signal in the object under consideration. This approach reveals at least three major problems. (i) The input signals must be controlled isotopically, which is almost impossible in many realistic cases like free ranging animals. (ii) Both equilibration periods may be very long, especially when the turnover rate of the object under consideration is very slow, which aggravates the first problem. (iii) The detection of small or slow pools is improved by large isotopic signal changes, but large isotopic changes also involve a considerable change in the input material; e.g. animal studies are usually carried out as diet-switch experiments, where the diet is switched between C3 and C4 plants, since C3 and C4 plants differ strongly in their isotopic signal. The

  19. Phosphatidylinositol 4-phosphate 5-kinase α contributes to Toll-like receptor 2-mediated immune responses in microglial cells stimulated with lipoteichoic acid.

    PubMed

    Nguyen, Tu Thi Ngoc; Seo, Eunjeong; Choi, Juyong; Le, Oanh Thi Tu; Kim, Ji Yun; Jou, Ilo; Lee, Sang Yoon

    2017-10-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important lipid regulator of membrane signaling and remodeling processes. Accumulating evidence indicates a link between PIP2 metabolism and Toll-like receptor (TLR) signaling, a key transducer of immune responses such as inflammation, phagocytosis, and autophagy. Microglia are immune effector cells that serve as macrophages in the brain. Here, we examined the potential role of phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα), a PIP2-producing enzyme, in TLR2 signaling in microglial cells. Treatment of BV2 microglial cells with lipoteichoic acid (LTA), a TLR2 agonist, increased PIP5Kα expression in BV2 and primary microglial cells, but not in primary cultures from TLR2-deficient mice. PIP5Kα knockdown of BV2 cells with shRNA significantly suppressed LTA-induced activation of TLR2 downstream signaling, including the production of proinflammatory cytokines and phosphorylation of NF-κB, JNK, and p38 MAP kinase. Such suppression was reversed by complementation of PIP5Kα. PIP5Kα knockdown lowered PIP2 levels and impaired LTA-induced plasma membrane targeting of TIRAP, a PIP2-dependent adaptor required for TLR2 activation. Besides, PIP5Kα knockdown inhibited phagocytic uptake of E. coli particles and autophagy-related vesicle formation triggered by LTA. Taken together, these results support that PIP5Kα can positively mediate TLR2-associated immune responses through PIP2 production in microglial cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Phosphatidylinositol- and phosphatidylcholine-transfer activity of PITPbeta is essential for COPI-mediated retrograde transport from the Golgi to the endoplasmic reticulum.

    PubMed

    Carvou, Nicolas; Holic, Roman; Li, Michelle; Futter, Clare; Skippen, Alison; Cockcroft, Shamshad

    2010-04-15

    Vesicles formed by the COPI complex function in retrograde transport from the Golgi to the endoplasmic reticulum (ER). Phosphatidylinositol transfer protein beta (PITPbeta), an essential protein that possesses phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) lipid transfer activity is known to localise to the Golgi and ER but its role in these membrane systems is not clear. To examine the function of PITPbeta at the Golgi-ER interface, RNA interference (RNAi) was used to knockdown PITPbeta protein expression in HeLa cells. Depletion of PITPbeta leads to a decrease in PtdIns(4)P levels, compaction of the Golgi complex and protection from brefeldin-A-mediated dispersal to the ER. Using specific transport assays, we show that anterograde traffic is unaffected but that KDEL-receptor-dependent retrograde traffic is inhibited. This phenotype can be rescued by expression of wild-type PITPbeta but not by mutants defective in docking, PtdIns transfer and PtdCho transfer. These data demonstrate that the PtdIns and PtdCho exchange activity of PITPbeta is essential for COPI-mediated retrograde transport from the Golgi to the ER.

  1. Phosphatidylinositol- and phosphatidylcholine-transfer activity of PITPβ is essential for COPI-mediated retrograde transport from the Golgi to the endoplasmic reticulum

    PubMed Central

    Carvou, Nicolas; Holic, Roman; Li, Michelle; Futter, Clare; Skippen, Alison; Cockcroft, Shamshad

    2010-01-01

    Vesicles formed by the COPI complex function in retrograde transport from the Golgi to the endoplasmic reticulum (ER). Phosphatidylinositol transfer protein β (PITPβ), an essential protein that possesses phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) lipid transfer activity is known to localise to the Golgi and ER but its role in these membrane systems is not clear. To examine the function of PITPβ at the Golgi-ER interface, RNA interference (RNAi) was used to knockdown PITPβ protein expression in HeLa cells. Depletion of PITPβ leads to a decrease in PtdIns(4)P levels, compaction of the Golgi complex and protection from brefeldin-A-mediated dispersal to the ER. Using specific transport assays, we show that anterograde traffic is unaffected but that KDEL-receptor-dependent retrograde traffic is inhibited. This phenotype can be rescued by expression of wild-type PITPβ but not by mutants defective in docking, PtdIns transfer and PtdCho transfer. These data demonstrate that the PtdIns and PtdCho exchange activity of PITPβ is essential for COPI-mediated retrograde transport from the Golgi to the ER. PMID:20332109

  2. Gliotoxin Suppresses Macrophage Immune Function by Subverting Phosphatidylinositol 3,4,5-Trisphosphate Homeostasis.

    PubMed

    Schlam, Daniel; Canton, Johnathan; Carreño, Marvin; Kopinski, Hannah; Freeman, Spencer A; Grinstein, Sergio; Fairn, Gregory D

    2016-04-05

    Aspergillus fumigatus, an opportunistic fungal pathogen, spreads in the environment by releasing numerous conidia that are capable of reaching the small alveolar airways of mammalian hosts. In otherwise healthy individuals, macrophages are responsible for rapidly phagocytosing and eliminating these conidia, effectively curbing their germination and consequent invasion of pulmonary tissue. However, under some circumstances, the fungus evades phagocyte-mediated immunity and persists in the respiratory tree. Here, we report thatA. fumigatusescapes macrophage recognition by strategically targeting phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] metabolism through gliotoxin, a potent immunosuppressive mycotoxin. Time-lapse microscopy revealed that, in response to the toxin, macrophages cease to ruffle, undergo abrupt membrane retraction, and fail to phagocytose large targets effectively. Gliotoxin was found to prevent integrin activation and interfere with actin dynamics, both of which are instrumental for phagocytosis; similar effects were noted in immortalized and primary phagocytes. Detailed studies of the underlying molecular mechanisms of toxicity revealed that inhibition of phagocytosis is attributable to impaired accumulation of PtdIns(3,4,5)P3and the associated dysregulation of downstream effectors, including Rac and/or Cdc42. Strikingly, in response to the diacylglycerol mimetic phorbol 12-myristate 13-acetate, gliotoxin-treated macrophages reactivate beta integrins, reestablish actin dynamics, and regain phagocytic capacity, despite the overt absence of plasmalemmal PtdIns(3,4,5)P3 Together, our findings identify phosphoinositide metabolism as a critical upstream target of gliotoxin and also indicate that increased diacylglycerol levels can bypass the requirement for PtdIns(3,4,5)P3signaling during membrane ruffling and phagocytosis. Aspergillus fumigatusis the most frequent cause of human infections in theAspergillusgenus. In immunocompromised

  3. Prevention of Proliferative Vitreoretinopathy by Suppression of Phosphatidylinositol 5-Phosphate 4-Kinases

    PubMed Central

    Ma, Gaoen; Duan, Yajian; Huang, Xionggao; Qian, Cynthia X.; Chee, Yewlin; Mukai, Shizuo; Cui, Jing; Samad, Arif; Matsubara, Joanne Aiko; Kazlauskas, Andrius; D'Amore, Patricia A.; Gu, Shuyan; Lei, Hetian

    2016-01-01

    Purpose Previous studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -β) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -β and whether suppression of PI5P4Kα and -β would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR). Methods PI5P4Kα and -β encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -β by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -β knocked-down ARPE-19 cells. Expression of PI5P4Kα and -β was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection. Results We found that vitreous enhanced expression of PI5P4Kα and -β in RPE cells and that knocking down PI5P4Kα and -β abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -β in the human PI5P4Kα and -β knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -β abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -β was abundant in epiretinal membranes from PVR grade C patients. Conclusions The findings from this study indicate that PI5P4Kα and -β could be novel therapeutic targets for the treatment of PVR. PMID:27472081

  4. Employee Development and Turnover Intention: Theory Validation

    ERIC Educational Resources Information Center

    Rahman, Wali; Nas, Zekeriya

    2013-01-01

    Purpose: This study aims to examine the pattern of behavior of turnover intentions in developing countries "vis-a-vis" the one in advanced countries through the empirical data from public universities in Khyber Pakhtunkhwa, Pakistan. The study provides empirical evidence from academia in Pakistan, thereby enriching the understanding of…

  5. Home Visitor Job Satisfaction and Turnover.

    ERIC Educational Resources Information Center

    Buchbinder, Sharon B.; Duggan, Anne K.; Young, Elizabeth; Fuddy, Loretta; Sia, Cal

    This paper summarizes findings of a 3-year study of the job satisfaction and turnover of home visitors, both professional and paraprofessional, in programs which link families-at-risk for impaired functioning to medical home care and other resources. Specifically, the study examined: (1) home visitor personal characteristics that influence…

  6. Antecedents of Norwegian Beginning Teachers' Turnover Intentions

    ERIC Educational Resources Information Center

    Tiplic, Dijana; Brandmo, Christian; Elstad, Eyvind

    2015-01-01

    This study aims at exploring several individual, organizational, and contextual factors that may affect beginning teachers' turnover intentions during their first years of practice. The sample consists of 227 beginning teachers (69% female and 31% male) from 133 schools in Norway. The results show four important antecedents of beginning teachers'…

  7. Health care workplace discrimination and physician turnover.

    PubMed

    Nunez-Smith, Marcella; Pilgrim, Nanlesta; Wynia, Matthew; Desai, Mayur M; Bright, Cedric; Krumholz, Harlan M; Bradley, Elizabeth H

    2009-12-01

    To examine the association between physician race/ ethnicity, workplace discrimination, and physician job turnover. Cross-sectional, national survey conducted in 2006-2007 of practicing physicians (n = 529) randomly identified via the American Medical Association Masterfile and the National Medical Association membership roster. We assessed the relationships between career racial/ethnic discrimination at work and several career-related dependent variables, including 2 measures of physician turnover, career satisfaction, and contemplation of career change. We used standard frequency analyses, odds ratios and chi2 statistics, and multivariate logistic regression modeling to evaluate these associations. Physicians who self-identified as nonmajority were significantly more likely to have left at least 1 job because of workplace discrimination (black, 29%; Asian, 24%; other race, 21%; Hispanic/Latino, 20%; white, 9%). In multivariate models, having experienced racial/ethnic discrimination at work was associated with high job turnover (adjusted odds ratio, 2.7; 95% CI, 1.4-4.9). Among physicians who experienced workplace discrimination, only 45% of physicians were satisfied with their careers (vs 88% among those who had not experienced workplace discrimination, p value < .01), and 40% were contemplating a career change (vs 10% among those who had not experienced workplace discrimination, p value < .001). Workplace discrimination is associated with physician job turnover, career dissatisfaction, and contemplation of career change. These findings underscore the importance of monitoring for workplace discrimination and responding when opportunities for intervention and retention still exist.

  8. Teacher Turnover in Charter Schools. Research Brief

    ERIC Educational Resources Information Center

    Stuit, David; Smith, Thomas M.

    2010-01-01

    The current study aimed to contribute to a deeper understanding of the organizational conditions of charter schools by examining teacher turnover. Using data from the National Center for Education Statistics (NCES) 2003-04 Schools and Staffing Survey (SASS) and the Teacher Follow-Up Survey (TFS), researchers from the National Center on School…

  9. Director Turnover: An Australian Academic Development Study

    ERIC Educational Resources Information Center

    Fraser, Kym; Ryan, Yoni

    2012-01-01

    Although it can be argued that directors of central academic development units (ADUs) are critical to the implementation of university teaching and learning strategies, it would appear there is a high director turnover rate. While research in the USA, the UK, and Australia illustrates that ADUs are frequently closed or restructured, that research…

  10. Costing Child Protective Services Staff Turnover.

    ERIC Educational Resources Information Center

    Graef, Michelle I.; Hill, Erick L.

    2000-01-01

    Details process of determining a child welfare agency's actual dollar costs directly attributed to protective services staff turnover, using the agency's human resources database and interviews with administrative personnel. Provides formulas and process for calculating specific cost elements due to employee separation, replacement, and training.…

  11. Director Turnover: An Australian Academic Development Study

    ERIC Educational Resources Information Center

    Fraser, Kym; Ryan, Yoni

    2012-01-01

    Although it can be argued that directors of central academic development units (ADUs) are critical to the implementation of university teaching and learning strategies, it would appear there is a high director turnover rate. While research in the USA, the UK, and Australia illustrates that ADUs are frequently closed or restructured, that research…

  12. Turnover of Public School Superintendents in Arizona

    ERIC Educational Resources Information Center

    Meyer, Joyce Ntsoaki

    2013-01-01

    This study used a descriptive qualitative design utilizing a phenomenological approach to determine and examine the reasons behind the voluntary or involuntary turnover of Arizona school superintendents. Open-ended questions were used to interview five superintendents who had left their districts between 2008 and 2013 about their perceptions on…

  13. Minor psychiatric morbidity and labour turnover.

    PubMed Central

    Jenkins, R

    1985-01-01

    The relation of minor psychiatric morbidity with labour turnover is examined, using data from a study of young, predominantly middle class, white collar men and women. The results suggest that the presence of psychiatric symptomatology is at least as important as occupational attitudes in identifying individuals who would subsequently leave the organisation. PMID:4016004

  14. Antecedents of Norwegian Beginning Teachers' Turnover Intentions

    ERIC Educational Resources Information Center

    Tiplic, Dijana; Brandmo, Christian; Elstad, Eyvind

    2015-01-01

    This study aims at exploring several individual, organizational, and contextual factors that may affect beginning teachers' turnover intentions during their first years of practice. The sample consists of 227 beginning teachers (69% female and 31% male) from 133 schools in Norway. The results show four important antecedents of beginning teachers'…

  15. Employee Development and Turnover Intention: Theory Validation

    ERIC Educational Resources Information Center

    Rahman, Wali; Nas, Zekeriya

    2013-01-01

    Purpose: This study aims to examine the pattern of behavior of turnover intentions in developing countries "vis-a-vis" the one in advanced countries through the empirical data from public universities in Khyber Pakhtunkhwa, Pakistan. The study provides empirical evidence from academia in Pakistan, thereby enriching the understanding of…

  16. Dynamics of telomeric DNA turnover in yeast.

    PubMed Central

    McEachern, Michael J; Underwood, Dana Hager; Blackburn, Elizabeth H

    2002-01-01

    Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover. PMID:11805045

  17. KCNE1-KCNQ1 osmoregulation by interaction of phosphatidylinositol-4,5-bisphosphate with Mg2+ and polyamines.

    PubMed

    Piron, Julien; Choveau, Frank S; Amarouch, Mohammed Yassine; Rodriguez, Nicolas; Charpentier, Flavien; Mérot, Jean; Baró, Isabelle; Loussouarn, Gildas

    2010-09-15

    KCNQ1 osmosensitivity is of physiological and pathophysiological relevance in epithelial and cardiac cells, but the mechanism involved remains elusive. In COS-7 cells expressing the KCNE1-KCNQ1 fusion protein, extracellular hypoosmolarity and hyperosmolarity modify the channel biophysical parameters. These changes are consistent with hypoosmolarity increasing the level of membrane phosphatidylinositol-4,5-bisphosphate (PIP(2)), which in turn upregulates KCNE1-KCNQ1 channels. We showed that increasing PIP(2) levels with a water-soluble PIP(2) analogue prevented channel upregulation in hypoosmotic condition, suggesting a variation of the channel-PIP(2) interaction during channel osmoregulation. Furthermore, we showed that polyamines and Mg(2+), already known to tonically inhibit KCNQ channels by screening PIP(2) negative charges, are involved in the osmoregulatory process. Indeed, intracellular Mg(2+) removal and polyamines chelation inhibited the channel osmoregulation. Thus, the dilution of those cations during cell swelling might decrease channel inhibition and explain the channel upregulation by hypoosmolarity. To support this idea, we quantified the role of Mg(2+) in the osmodependent channel activity. Direct measurement of intracellular [Mg(2+)] variations during osmotic changes and characterization of the channel Mg(2+) sensitivity showed that Mg(2+) participates significantly to the osmoregulation. Using intracellular solutions that mimic the variation of Mg(2+) and polyamines, we were able to recapitulate the current amplitude variations in response to extracellular osmolarity changes. Altogether, these results support the idea of a modulation of the channel-PIP(2) interactions by Mg(2+) and polyamines during cell volume changes. It is likely that this mechanism applies to other channels that are sensitive to both osmolarity and PIP(2).

  18. Phosphatidylinositol 3-kinase-dependent signaling modulates taurochenodeoxycholic acid-induced liver injury and cholestasis in perfused rat livers.

    PubMed

    Rust, Christian; Bauchmuller, Kris; Fickert, Peter; Fuchsbichler, Andrea; Beuers, Ulrich

    2005-07-01

    Taurochenodeoxycholic acid (TCDCA), but not glycochenodeoxycholic acid (GCDCA), activates a phosphatidylinositol 3-kinase (PI3-K)-mediated survival pathway in vitro. Here, the effects of PI3-K inhibition on TCDCA- and GCDCA-induced hepatocellular injury, apoptosis, and bile secretion were examined in the intact liver. In isolated perfused rat livers, bile flow was determined gravimetrically. Hepatovenous lactate dehydrogenase and alanine aminotransferase efflux as markers of liver integrity and biliary secretion of 2,4-dinitrophenyl-S-glutathione (DNP-GS) were determined photometrically. Apoptosis was assessed by immunohistochemistry of active caspase-3 and cytokeratin 18 in liver tissue. Phosphorylation of protein kinase B (PKB/Akt) as a readout of PI3-K activity was determined by immunoblot analysis. Bile acid concentrations were determined by gas chromatography. TCDCA (25 muM) induced moderate liver injury by hepatocellular apoptosis and distinctly reduced bile flow and DNP-GS secretion. In contrast, GCDCA (25 muM) induced severe liver injury by extensive hepatocyte apoptosis. TCDCA strongly activated PI3-K, whereas GCDCA did not markedly affect PI3-K activity. Inhibition of PI3-K by 100 nM wortmannin enhanced TCDCA-induced liver injury and apoptosis and tended to aggravate the cholestatic effect of TCDCA. In contrast, wortmannin reduced GCDCA-induced liver injury and apoptosis. Bile acid uptake tended to be reduced by wortmannin. The cholestatic effect of GCDCA was aggravated by wortmannin. Inhibition of PI3-K markedly aggravated TCDCA-induced but not GCDCA-induced liver damage and hepatocyte apoptosis. Thus TCDCA appears to block its inherent toxicity by a PI3-K-dependent survival pathway in the intact liver.

  19. H1047R phosphatidylinositol 3-kinase mutant enhances HER2-mediated transformation via heregulin production and activation of HER3

    PubMed Central

    Chakrabarty, Anindita; Rexer, Brent N.; Wang, Shizhen Emily; Cook, Rebecca S.; Engelman, Jeffrey A.; Arteaga, Carlos, L.

    2010-01-01

    Hyperactivation of phosphatidylinositol-3 kinase (PI3K) can occur as a result of somatic mutations in PIK3CA, the gene encoding the p110α subunit of PI3K. The HER2 oncogene is amplified in 25% of all breast cancers and some of these tumors also harbor PIK3CA mutations. We examined mechanisms by which mutant PI3K can enhance transformation and confer resistance to HER2-directed therapies. We introduced the PI3K mutations E545K and H1047R in MCF10A human mammary epithelial cells that also overexpress HER2. Both mutants conferred a gain of function to MCF10A/HER2 cells. Expression of H1047R PI3K but not E545K PI3K markedly upregulated the HER3/HER4 ligand heregulin (HRG). HRG siRNA inhibited growth of H1047R but not E545K-expressing cells and synergized with the HER2 inhibitors trastuzumab and lapatinib. The PI3K inhibitor BEZ235 markedly inhibited HRG and pAKT levels and, in combination with lapatinib, completely inhibited growth of cells expressing H1047R PI3K. These observations suggest that PI3K mutants enhance HER2-mediated transformation by amplifying the ligand-induced signaling output of the ErbB network. This also counteracts the full effect of therapeutic inhibitors of HER2. These data also suggest that mammary tumors that contain both HER2 gene amplification and PIK3CA mutations should be treated with a combination of HER2 and PI3K inhibitors. PMID:20581867

  20. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3β-dependent Cell–Cell Mechanical Adhesion

    PubMed Central

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1−/− mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and β-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated β-catenin through activation of GSK3β. Expression of a nondegradable form of β-catenin in PC-1 MDCK cells restores strong cell–cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

  1. Predicting nursing turnover with catastrophe theory.

    PubMed

    Wagner, Cheryl M

    2010-09-01

    This paper is a report of a study comparing an innovative nonlinear model and a traditional linear model for accuracy in prediction of nursing turnover. An international, sustained nursing shortage creates a need to target accurately the staff population at risk for turnover. Existing linear methodology is cumbersome with the number of variables needed, while producing inadequate results. Nonlinear modelling methods offer increased simplicity and accuracy in predictability. A correlational survey with a longitudinal cohort prospective study was carried out in 2005-2006 with a convenience sample of 1033 Registered Nurses from the Midwest region of the United States of America. At time 1, 756 usable questionnaires were returned and 496 at time 2. Data analysis included analyses of a cusp catastrophe model, a cube-shaped four-dimensional figure with a top that provided a down-turning slope area (the catastrophe/cusp zone). This fluid, dynamic cusp version employed the smallest number of control and dependent variables. The exceedingly small turnover sample preempted the use of the computerized program Cuspfit; a proven quasi-quantitative methodology demonstrated 80.4% predictability in the cusp catastrophe model overall and 53.6% correct predictions of actual terminations, particularly in nurses with <5 years of nursing experience. Additional accurate predictions were obtained with the use of a time-staged model. Organizational commitment and anticipated turnover were accurate predictor variables; job tension was not. Catastrophe models are useful in predicting nursing turnover. Future nursing researchers should act on this evidence to benefit forthcoming studies and the profession.

  2. Work-Related Variables and Turnover Intention among Registered Nurses.

    ERIC Educational Resources Information Center

    Pooyan, Abdullah; And Others

    1990-01-01

    Health institutions have become more interested in the causes of job turnover among registered nurses. Proper management of job turnover can improve the financial health and long-term survival of health care institutions. (Author)

  3. Staff turnover: occasional friend, frequent foe, and continuing frustration.

    PubMed

    McConnell, C R

    1999-09-01

    Turnover appears to be a relatively simple concept. However, considerable confusion results when discussing turnover because of differences in how it is defined--what is counted, how it is counted, and how the rate of turnover is expressed. Turnover is also costly, although not enough attention is paid to turnover's cost because so much of it is indirect and thus not readily visible. There are a variety of causes of turnover, some which can be corrected and some which cannot be avoided. Reducing or otherwise controlling turnover requires continuing management attention to its causes and constant recognition of what can and should be controlled and what cannot be controlled. Ongoing attention to turnover is an essential part of the department manager's role.

  4. Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein

    PubMed Central

    Kim, Kyoungtae; McCully, Michelle E.; Bhattacharya, Nandini; Butler, Boyd; Sept, David; Cooper, John A.

    2008-01-01

    The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP2). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP2 binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP2 binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP2 rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can “wobble” when bound to the barbed end solely by the C-terminal “tentacle” of its β-subunit. PMID:17182619

  5. Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

    PubMed Central

    ZHANG, Zong-Kang; LI, Jie; LIU, Jin; GUO, Baosheng; LEUNG, Albert; ZHANG, Ge; ZHANG, Bao-Ting

    2016-01-01

    Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment could significantly attenuate the decreases in the phosphorylation level of PI3K at p110 catalytic subunit and its downstream markers related to protein synthesis, and inhibit the increases in protein degradation markers at mRNA and protein levels in rat soleus muscle following 28-day hindlimb unloading. In addition, the decreases in soleus muscle mass, muscle fiber cross-sectional area, twitch force, specific force, contraction time and half relaxation time could be significantly attenuated by the high dose ICT treatment. The low dose ICT treatment could moderately attenuate the above changes induced by unloading. Wortmannin, a specific inhibitor of PI3K at p110 catalytic subunit, could abolish the above effects of ICT in vitro and in vivo, indicating that PI3K/Akt signaling could be required by ICT to counteract skeletal muscle atrophy following mechanical unloading. PMID:26831566

  6. Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci*

    PubMed Central

    Popkie, Anthony P.; Zeidner, Leigh C.; Albrecht, Ashley M.; D'Ippolito, Anthony; Eckardt, Sigrid; Newsom, David E.; Groden, Joanna; Doble, Bradley W.; Aronow, Bruce; McLaughlin, K. John; White, Peter; Phiel, Christopher J.

    2010-01-01

    Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. PMID:21047779

  7. Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

    SciTech Connect

    Kakuk, Annamaria; Friedlaender, Elza; Vereb, Gyoergy; Lisboa, Duarte; Bagossi, Peter; Toth, Gabor; Gergely, Pal; Vereb, Gyoergy

    2008-08-01

    PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence {sup 916}NFNHIHKRIRRVADKYLSG{sup 934} comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin {alpha}/{beta} mechanism employing importins {alpha}1 and {alpha}3 but not {alpha}5. This transport was inhibited by wheat germ agglutinin and GTP{gamma}S. The sequence {sup 1414}SKKTNRGSQLHKYYMKRRTL{sup 1433}, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin {alpha}1/{beta} or {alpha}3/{beta} complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

  8. Hepatitis C virus NS5A protein cooperates with phosphatidylinositol 4-kinase IIIα to induce mitochondrial fragmentation

    PubMed Central

    Siu, Gavin Ka Yu; Zhou, Fan; Yu, Mei Kuen; Zhang, Leiliang; Wang, Tuanlao; Liang, Yongheng; Chen, Yangchao; Chan, Hsiao Chang; Yu, Sidney

    2016-01-01

    Hepatitis C virus (HCV) has long been observed to take advantage of the host mitochondria to support viral replication and assembly. The HCV core protein has been implicated to fragment host mitochondria. In this report, we have discovered that the non-structural protein 5A (NS5A) plays an instructive role in attaching ER with mitochondria, causing mitochondrial fragmentation. Dynamin-related protein 1(Drp1), a host protein essential to mitochondrial membrane fission, does not play a role in NS5A-induced mitochondrial fragmentation. Instead, phosphatidylinositol 4-kinase IIIα (PI4KA), which has been demonstrated to bind to NS5A and is required to support HCV life cycle, is required for NS5A to induce mitochondrial fragmentation. Both NS5A and core are required by HCV to fragment the mitochondria, as inhibiting either of their respective downstream proteins, PI4KA or Drp1, resulted in lengthening of mitochondria tubules in HCVcc-infected cells. By fragmenting the mitochondria, NS5A renders the cells more resistant to mitochondria mediated apoptosis. This finding indicates previously-ignored contribution of NS5A in HCV-induced mitochondria dysfunction. PMID:27010100

  9. Phosphatidylinositol 3-kinase and calcium-activated transcription pathways are required for VLDL-induced smooth muscle cell proliferation.

    PubMed

    Lipskaia, Larissa; Pourci, Marie-Luce; Deloménie, Claudine; Combettes, Laurent; Goudounèche, Dominique; Paul, Jean-Louis; Capiod, Thierry; Lompré, Anne-Marie

    2003-05-30

    Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca2+ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose- and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca2+ release was inhibited and the IP3-sensitive Ca2+ release induced by ATP (10 micromol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3beta in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca2+-induced Ca2+ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype.

  10. Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.

    PubMed

    Martelli, A M; Evangelisti, C; Follo, M Y; Ramazzotti, G; Fini, M; Giardino, R; Manzoli, L; McCubrey, J A; Cocco, L

    2011-01-01

    Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.

  11. Functional control of cold- and menthol-sensitive TRPM8 ion channels by phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Liu, Beiying; Qin, Feng

    2005-02-16

    Cold is detected by a small subpopulation of peripheral thermoreceptors. TRPM8, a cloned menthol- and cold-sensitive ion channel, has been suggested to mediate cold transduction in the innocuous range. The channel shows a robust response in whole-cell recordings but exhibits markedly reduced activity in excised membrane patches. Here we report that phosphatidylinositol 4,5-bisphosphate (PIP2) is an essential regulator of the channel function. The rundown of the channel is prevented by lipid phosphatase inhibitors. Application of exogenous PIP2 both activates the channel directly and restores rundown activity. Whole-cell experiments involving intracellular dialysis with polyvalent cations, inhibition of PIP2 synthesis kinases, and receptor-mediated hydrolysis of PIP2 show that PIP2 also modulates the channel activity in the intact cells. The crucial role of PIP2 on the function of TRPM8 suggests that the membrane PIP2 level may be an important regulator of cold transduction in vivo. The opposite effects of PIP2 on the vanilloid receptor TRPV1 and TRPM8 also implies that the membrane lipid may have dual actions as a bimodal switch to selectively control the heat- and cold-induced responses in nociceptors expressing both channels.

  12. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

    PubMed

    Vicente-Manzanares, M; Rey, M; Jones, D R; Sancho, D; Mellado, M; Rodriguez-Frade, J M; del Pozo, M A; Yáñez-Mó, M; de Ana, A M; Martínez-A, C; Mérida, I; Sánchez-Madrid, F

    1999-10-01

    The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

  13. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration.

    PubMed

    Dise, Rebecca S; Frey, Mark R; Whitehead, Robert H; Polk, D Brent

    2008-01-01

    Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.

  14. Salary and Ranking and Teacher Turnover: A Statewide Study

    ERIC Educational Resources Information Center

    Garcia, Cynthia Martinez; Slate, John R.; Delgado, Carmen Tejeda

    2009-01-01

    This study examined three years of data obtained from the Academic Excellence Indicator System of the State of Texas regarding teacher turnover rate and teacher salary. Across all public school districts, teacher salary was consistently negatively related to teacher turnover; that is, where salary was lower, turnover rate was higher When data were…

  15. Turnover of regulated nurses in long-term care facilities.

    PubMed

    Chu, Charlene H; Wodchis, Walter P; McGilton, Katherine S

    2014-07-01

    To describe the relationship between nursing staff turnover in long-term care (LTC) homes and organisational factors consisting of leadership practices and behaviours, supervisory support, burnout, job satisfaction and work environment satisfaction. The turnover of regulated nursing staff [Registered Nurses (RNs) and Registered Practical Nurses (RPNs)] in LTC facilities is a pervasive problem, but there is a scarcity of research examining this issue in Canada. The study was conceptualized using a Stress Process model. Distinct surveys were distributed to administrators to measure organisational factors and to regulated nurses to measure personal and job-related sources of stress and workplace support. In total, 324 surveys were used in the linear regression analysis to examine factors associated with high turnover rates. Higher leadership practice scores were associated with lower nursing turnover; a one score increase in leadership correlated with a 49% decrease in nursing turnover. A significant inverse relationship between leadership turnover and nurse turnover was found: the higher the administrator turnover the lower the nurse turnover rate. Leadership practices and administrator turnover are significant in influencing regulated nurse turnover in LTC. Long-term care facilities may want to focus on building good leadership and communication as an upstream method to minimize nurse turnover. © 2013 John Wiley & Sons Ltd.

  16. The shocking cost of turnover in health care.

    PubMed

    Waldman, J Deane; Kelly, Frank; Arora, Sanjeev; Smith, Howard L

    2004-01-01

    Review of turnover costs at a major medical center helps health care managers gain insights about the magnitude and determinants of this managerial challenge and assess the implications for organizational effectiveness. Here, turnover includes hiring, training, and productivity loss costs. Minimum cost of turnover represented a loss of >5 percent of the total annual operating budget.

  17. Factors Related to Turnover among Mental Health Workers.

    ERIC Educational Resources Information Center

    Tang, Thomas Li-Ping

    In view of the extremely high turnover among corporation recruits, there is growing and justified interest in having organizations identify the causes of turnover and possible ways of reducing it. Many studies have examined different variables related to turnover, including organizational commitment, career commitment, job satisfaction, and…

  18. 41 CFR 109-27.5002 - Stores inventory turnover ratio.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... turnover ratio. 109-27.5002 Section 109-27.5002 Public Contracts and Property Management Federal Property....5002 Stores inventory turnover ratio. Comparison of investment in stores inventories to annual issues... comparison may be expressed either as a turnover ratio (dollar value of issues divided by dollar value...

  19. 41 CFR 109-27.5002 - Stores inventory turnover ratio.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... turnover ratio. 109-27.5002 Section 109-27.5002 Public Contracts and Property Management Federal Property....5002 Stores inventory turnover ratio. Comparison of investment in stores inventories to annual issues... comparison may be expressed either as a turnover ratio (dollar value of issues divided by dollar value...

  20. 41 CFR 109-27.5002 - Stores inventory turnover ratio.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... turnover ratio. 109-27.5002 Section 109-27.5002 Public Contracts and Property Management Federal Property....5002 Stores inventory turnover ratio. Comparison of investment in stores inventories to annual issues... comparison may be expressed either as a turnover ratio (dollar value of issues divided by dollar value...

  1. 41 CFR 109-27.5002 - Stores inventory turnover ratio.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... turnover ratio. 109-27.5002 Section 109-27.5002 Public Contracts and Property Management Federal Property....5002 Stores inventory turnover ratio. Comparison of investment in stores inventories to annual issues... comparison may be expressed either as a turnover ratio (dollar value of issues divided by dollar value...

  2. 41 CFR 109-27.5002 - Stores inventory turnover ratio.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... turnover ratio. 109-27.5002 Section 109-27.5002 Public Contracts and Property Management Federal Property....5002 Stores inventory turnover ratio. Comparison of investment in stores inventories to annual issues... comparison may be expressed either as a turnover ratio (dollar value of issues divided by dollar value...

  3. Superintendent Turnover in Kentucky. Issues & Answers. REL 2011-No. 113

    ERIC Educational Resources Information Center

    Johnson, Jerry; Huffman, Tyler; Madden, Karen; Shope, Shane

    2011-01-01

    This study examines superintendent turnover in Kentucky public school districts for 1998/99-2007/08, looking at how turnover varies by rural status, Appalachian and non-Appalachian region, and 2007/08 school district characteristics. Key findings include: (1) Kentucky school districts averaged one superintendent turnover during 1998/99-2007/08;…

  4. Organ-specific changes in norepinephrine turnover against various stress conditions in thermoneutral mice.

    PubMed

    Teramura, Yasufumi; Terao, Akira; Okada, Yuko; Tomida, Junichi; Okamatsu-Ogura, Yuko; Kimura, Kazuhiro

    2014-08-01

    The effects of three stressors of different categories, namely cold exposure, immobilization, and lipopolysaccharide (LPS) treatment, on sympathetic nerve activity were examined by assessing its biochemical index norepinephrine (NE) turnover in peripheral organs of C57BL/6 mice. NE turnover was assessed by measuring the decrease in the organ NE concentration 3 h after inhibition of catecholamine biosynthesis with alpha-methyl-p-tyrosine. NE turnover in brown adipose tissue (BAT) in the room temperature (23 degrees C) control group was as high as that in the cold exposure (4 degrees C) group. Similarly, the mRNA level of the thermogenic marker uncoupling protein 1 (UCP1) in the room temperature control group was as high as that in the cold exposure group. As sympathetic stimulation upregulates the UCP1 mRNA level, we thought that sympathetic nerve tonus in BAT was already accelerated at room temperature. To exclude factors affecting basal sympathetic nerve activity, mice housed at thermoneutral temperature (30 degrees C) were used as controls for the subsequent experiments. In this condition, cold exposure accelerated NE turnover in the BAT, as well as heart and pancreas. The corticosterone level showed a higher trend in the cold exposure group in comparison to the control group. Immobilization accelerated NE turnover in the spleen, pancreas, and white adipose tissue and elevated the corticosterone level. LPS (3 mg/kg, i.p.) did not affect NE turnover in all peripheral organs but elevated the corticosterone level. In summary, the sympathetic nervous and adrenocortical responses to three stressors differed greatly. In particular, sympathetic responses showed clear organ-specific acceleration patterns. This important feature may improve our understanding of the multiplicity of biological responses.

  5. Norepinephrine turnover in the goldfish brain is modulated by sex steroids and GABA.

    PubMed

    Trudeau, V L; Sloley, B D; Peter, R E

    1993-10-08

    It is known that norepinephrine (NE) is important in the neuroendocrine control of pituitary gonadotropin II (GTH-II) and growth hormone (GH) release but very little is known about the factors regulating NE neurons in the goldfish brain. Female gonad-intact goldfish were implanted intraperitoneally (100 micrograms/g) with testosterone (T) or estradiol (E2) to elevate serum steroid levels. High-performance liquid chromatography measurements showed that steroid implantation had no effect on NE content in the telencephalon, including preoptic area (TEL-POA), or the hypothalamus (HYP). The turnover rate of NE was estimated from the rate of depletion of NE content from tissues following inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (240 micrograms/g). The present study demonstrates that E2 can decrease NE turnover rates in TEL-POA and HYP of sexually regressed goldfish (August). The results in recrudescent fish (November), however, indicate a more complex interaction of E2 with NE neurons since E2 increased NE turnover in TEL-POA and HYP in these animals. Testosterone (T) has less prominent effects on NE turnover rates in TEL-POA and HYP; the only significant effect of T-implantation was a small reduction of NE turnover in the TEL-POA of sexually recrudescent fish. Elevation of endogenous brain GABA concentrations by injection of the GABA transaminase inhibitor, gamma-vinyl-GABA (300 micrograms/g), significantly reduced NE turnover in TEL-POA. These data demonstrate that goldfish NE neurons in the TEL-POA are sensitive to regulation by changes in circulating sex steroids and by increases in brain GABA.

  6. Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and ubiquitin ligase expression in rainbow trout primary myocytes.

    PubMed

    Cleveland, Beth M; Weber, Gregory M

    2010-02-01

    The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of 4-day-old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 14% (P < 0.05). Treatment with 1 microM insulin increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 17% (P < 0.05). Supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine did not increase protein synthesis rates but reduced rates of protein degradation by 8% (P < 0.05). IGF-I (1 nM-100 nM) and insulin (1 nM-1 microM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of approximately 70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P < 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 (P < 0.05), which was inhibited by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10-20% (P < 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% (P < 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation decreased rates of protein synthesis by 60% (P < 0.05), increased protein degradation by 40% (P < 0.05), and increased expression of all ubiquitin ligases. These data suggest that, similar to mammals, the inhibitory effects of IGF-I and insulin on proteolysis occur via P I3-kinase/protein kinase B signaling and are partially responsible for the ability of these compounds to promote protein accretion.

  7. Histones cause aggregation and fusion of lipid vesicles containing phosphatidylinositol-4-phosphate.

    PubMed

    Lete, Marta G; Sot, Jesus; Gil, David; Valle, Mikel; Medina, Milagros; Goñi, Felix M; Alonso, Alicia

    2015-02-17

    In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle.

  8. Histones Cause Aggregation and Fusion of Lipid Vesicles Containing Phosphatidylinositol-4-Phosphate

    PubMed Central

    Lete, Marta G.; Sot, Jesus; Gil, David; Valle, Mikel; Medina, Milagros; Goñi, Felix M.; Alonso, Alicia

    2015-01-01

    In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle. PMID:25692591

  9. Lupus anticoagulant activities of murine monoclonal antibodies to liposomal phosphatidylinositol phosphate.

    PubMed

    Alving, B M; Banerji, B; Fogler, W E; Alving, C R

    1987-08-01

    Four murine monoclonal antibodies having high levels of activity against phosphatidyl-inositol phosphate (PIP) were tested for lupus anticoagulant activity. The antibodies showed different degrees of potency in a modified partial thromboplastin time test (APTT) that used dilutions of either bovine brain extract (Thrombofax) or liposomes consisting of phosphatidylcholine/phosphatidylserine (PC/PS) as the phospholipid source. The same relative order of anticoagulant potency that was observed in the APTT that used the PC/PS liposomes was maintained when the anti-PIP antibodies were tested for cross-reactivity either by induction of complement-dependent immune damage to liposomes containing PS, or in enzyme-linked immunosorbent assays that used PS, cardiolipin (CL), or phosphatidylinositol (PI) as antigens. The data indicate that monoclonal antibodies to PIP can express anticoagulant activity in a modified APTT that correlates with their different degrees of cross-reactivity against the negatively-charged phospholipids PS, CL, and PI.

  10. Lupus anticoagulant activities of murine monoclonal antibodies to liposomal phosphatidylinositol phosphate.

    PubMed Central

    Alving, B M; Banerji, B; Fogler, W E; Alving, C R

    1987-01-01

    Four murine monoclonal antibodies having high levels of activity against phosphatidyl-inositol phosphate (PIP) were tested for lupus anticoagulant activity. The antibodies showed different degrees of potency in a modified partial thromboplastin time test (APTT) that used dilutions of either bovine brain extract (Thrombofax) or liposomes consisting of phosphatidylcholine/phosphatidylserine (PC/PS) as the phospholipid source. The same relative order of anticoagulant potency that was observed in the APTT that used the PC/PS liposomes was maintained when the anti-PIP antibodies were tested for cross-reactivity either by induction of complement-dependent immune damage to liposomes containing PS, or in enzyme-linked immunosorbent assays that used PS, cardiolipin (CL), or phosphatidylinositol (PI) as antigens. The data indicate that monoclonal antibodies to PIP can express anticoagulant activity in a modified APTT that correlates with their different degrees of cross-reactivity against the negatively-charged phospholipids PS, CL, and PI. PMID:2820640

  11. Orthophosphate turnover in East African lakes.

    PubMed

    Peters, Robert Henry; MacIntyre, Sally

    1976-12-01

    Turnover rates of (32)P-PO4 and concentrations of orthophosphate as soluble reactive phosphorus (SRP) were measured in five East African waters. Rapid incorporation of (32)P-PO4 by the seston and orthophosphate concentrations below the limit of detectibility were found in Lakes Elmenteita, Naivasha, and Naivasha Crater Lake. Turnover was slow and orthophosphate concentration high in both Lake Nakuru and the Crescent Island Crater basin of Lake Naivasha. Further experiments in Lake Nakuru indicated that colloidal binding of orthophosphate was limited and that particles retained by an 8.0 μ filter incorporated 66% as much tracer as particles retained by a 0.1 μ filter. These experiments strengthen our conclusion that a large quantity of orthophosphate is available for algal use in Lake Nakuru.

  12. Replicator dynamics with turnover of players

    NASA Astrophysics Data System (ADS)

    Juul, Jeppe; Kianercy, Ardeshir; Bernhardsson, Sebastian; Pigolotti, Simone

    2013-08-01

    We study adaptive dynamics in games where players abandon the population at a given rate and are replaced by naive players characterized by a prior distribution over the admitted strategies. We demonstrate how such a process leads macroscopically to a variant of the replicator equation, with an additional term accounting for player turnover. We study how Nash equilibria and the dynamics of the system are modified by this additional term for prototypical examples such as the rock-paper-scissors game and different classes of two-action games played between two distinct populations. We conclude by showing how player turnover can account for nontrivial departures from Nash equilibria observed in data from lowest unique bid auctions.

  13. Occupational turnover intentions among substance abuse counselors

    PubMed Central

    Rothrauff, Tanja C.; Abraham, Amanda J.; Bride, Brian E.; Roman, Paul M.

    2010-01-01

    This study examined predictor, moderator, and mediator variables of occupational turnover intention (OcTI) among substance abuse counselors. Data were obtained via questionnaires from 929 counselors working in 225 private substance abuse treatment (SAT) programs across the U.S. Hierarchical multiple regression models were conducted to assess predictor, moderator, and mediator variables of OcTI. OcTI scores were relatively low on a 7-point scale, indicating that very few counselors definitely intended to leave the SAT field. Age, certification, positive perceptions of procedural and distributive justice, and hospital-based status negatively predicted OcTI. Counselors’ substance use disorder impacted history moderated the association between organizational commitment and OcTI. Organizational turnover intention partially mediated the link between organizational commitment and OcTI. Workforce stability might be achieved by promoting perceptions of advantages to working in a particular treatment program, organizational commitment, showing appreciation for counselors’ work, and valuing employees from diverse backgrounds. PMID:20947285

  14. Turnover intention in new graduate nurses: a multivariate analysis

    PubMed Central

    Beecroft, Pauline C; Dorey, Frederick; Wenten, Madé

    2008-01-01

    Title Turnover intention in new graduate nurses: a multivariate analysis Aim This paper is a report of a study to determine the relationship of new nurse turnover intent with individual characteristics, work environment variables and organizational factors and to compare new nurse turnover with actual turnover in the 18 months of employment following completion of a residency. Background Because of their influence on patient safety and health outcomes nurse turnover and turnover intent have received considerable attention worldwide. When nurse staffing is inadequate, especially during nursing shortages, unfavourable clinical outcomes have been documented. Method Prospective data collection took place from 1999 to 2006 with 889 new paediatric nurses who completed the same residency. Scores on study instruments were related to likelihood of turnover intent using logistic regression analysis models. Relationships between turnover intent and actual turnover were compared using Kaplan–Meier survivorship. Results The final model demonstrated that older respondents were more likely to have turnover intent if they did not get their ward choice. Also higher scores on work environment and organizational characteristics contributed to likelihood that the new nurse would not be in the turnover intent group. These factors distinguish a new nurse with turnover intent from one without 79% of the time. Increased seeking of social support was related to turnover intent and older new graduates were more likely to be in the turnover intent group if they did not get their ward choice. Conclusion When new graduate nurses are satisfied with their jobs and pay and feel committed to the organization, the odds against turnover intent decrease. What is already known about this topic There is concern in many countries about nurse turnover and the resulting effects on patient safety and quality of care. Decreasing ability to recruit experienced nurses has increased the emphasis on

  15. Review of nursing turnover research, 1977-1996.

    PubMed

    Tai, T W; Bame, S I; Robinson, C D

    1998-12-01

    Turnover represents a major problem for health care services in terms of cost and quality of care given. As a result, turnover has been the subject of a large number of investigations. However, the variety of study populations, research methodologies, and inconsistent definitions and measurements of turnover lead to difficulties when attempting to compare studies. The purpose of this paper is to present: (1) a summary of turnover study methods and procedures, and (2) a summary of socio-demographic, organizational, and social support factors associated with turnover of nursing staff.

  16. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    SciTech Connect

    Kouchi, Zen; Fujiwara, Yuki; Yamaguchi, Hideki; Nakamura, Yoshikazu; Fukami, Kiyoko

    2011-05-20

    Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

  17. Health Care Workplace Discrimination and Physician Turnover

    PubMed Central

    Nunez-Smith, Marcella; Pilgrim, Nanlesta; Wynia, Matthew; Desai, Mayur M.; Bright, Cedric; Krumholz, Harlan M.; Bradley, Elizabeth H.

    2013-01-01

    Objective To examine the association between physician race/ethnicity, workplace discrimination, and physician job turnover. Methods Cross-sectional, national survey conducted in 2006–2007 of practicing physicians [n = 529] randomly identified via the American Medical Association Masterfile and The National Medical Association membership roster. We assessed the relationships between career racial/ethnic discrimination at work and several career-related dependent variables, including 2 measures of physician turnover, career satisfaction, and contemplation of career change. We used standard frequency analyses, odds ratios and χ2 statistics, and multivariate logistic regression modeling to evaluate these associations. Results Physicians who self-identified as nonmajority were significantly more likely to have left at least 1 job because of workplace discrimination (black, 29%; Asian, 24%; other race, 21%; Hispanic/Latino, 20%; white, 9%). In multivariate models, having experienced racial/ethnic discrimination at work was associated with high job turnover [adjusted odes ratio, 2.7; 95% CI, 1.4–4.9]. Among physicians who experienced work-place discrimination, only 45% of physicians were satisfied with their careers (vs 88% among those who had not experienced workplace discrimination, p value < .01], and 40% were con-templating a career change (vs 10% among those who had not experienced workplace discrimination, p value < .001). Conclusion Workplace discrimination is associated with physician job turnover, career dissatisfaction, and contemplation of career change. These findings underscore the importance of monitoring for workplace discrimination and responding when opportunities for intervention and retention still exist. PMID:20070016

  18. Turnover of soil monosaccharides: Recycling versus Stabilization

    NASA Astrophysics Data System (ADS)

    Basler, Anna; Dyckmans, Jens

    2014-05-01

    Soil organic matter (SOM) represents a mixture of differently degradable compounds. Each of these compounds are characterised by different dynamics due to different chemical recalcitrance, transformation or stabilisation processes in soil. Carbohydrates represent one of these compounds and contribute up to 25 % to the soil organic matter. Vascular plants are the main source of pentose sugars (Arabinose and Xylose), whereas hexoses (Galactose and Mannose) are primarily produced by microorganisms. Several studies suggest that the mean turnover times of the carbon in soil sugars are similar to the turnover dynamics of the bulk carbon in soil. The aim of the study is to characterise the influence of stabilisation and turnover of soil carbohydrates. Soil samples are collected from (i) a continuous maize cropping experiment ('Höhere Landbauschule' Rotthalmünster, Bavaria) established 1979 on a Stagnic Luvisol and (ii) from a continuous wheat cropping, established 1969, as reference site. The effect of stabilisation is estimated by the comparison of turnover times of microbial and plant derived soil carbohydrates. As the dynamics of plant derived carbohydrate are solely influenced by stabilisation processes, whereas the dynamics of microbial derived carbohydrates are affected by recycling of organic carbon compounds derived by C3 plant substrate as well as stabilisation processes. The compound specific isotopic analysis (CSIA) of soil carbohydrates was performed using a HPLC/o/IRMS system. The chromatographic and mass spectrometric subunits were coupled with a LC-Isolink interface. Soil sugars were extracted after mild hydrolysis using 4 M trifluoroacetic acid (TFA). Chromatographic separation of the sugars was achieved using a low strength 0.25 mM NaOH solution as mobile phase at a ?ow rate of 250 μL min-1 at 10 ° C.

  19. Costing child protective services staff turnover.

    PubMed

    Graef, M I; Hill, E L

    2000-01-01

    This article details the process used in one state to determine the financial costs to the child welfare agency accrued over the course of one year that were directly attributable to CPS staff turnover. The formulas and process for calculating specific cost elements due to separation, replacement and training are provided. The practical considerations inherent in this type of analysis are highlighted, as well as the use of this type of data to inform agency human resource strategies.

  20. Water turnover assessment in overweight adolescents.

    PubMed

    O'Connell, Bláthnaid N; Weinheimer, Eileen M; Martin, Berdine R; Weaver, Connie M; Campbell, Wayne W

    2011-02-01

    Adequate intake (AI) standards for water in adolescents range between 2.4-3.3 l/day for males and 2.1-2.3 l/day for females, independent of obesity status. Water intakes and excretions of this population are not well documented. The purposes of this study were to assess water turnover, inputs, and outputs in overweight adolescents, compare these parameters between males and females, and evaluate the reproducibility of water turnover. Eighteen girls (BMI 31.7 ± 4 kg/m(2); mean ± s.d.) and nine boys (BMI 26.3 ± 3 kg/m(2)) aged 12-15 years completed two 3-week metabolic balance trials. Rate of water turnover (rH(2)O) was measured by tracking the decline of deuterated water from the body over 14 days. Water inputs (diet*, ad libitum(#), metabolic(#)) and outputs (urine*, feces*, insensible(#)) were assessed (*measured, #estimated). rH(2)O was lower (P = 0.002) in girls vs. boys (3,742 ± 536 vs. 4,537 ± 623 g/day). Per kg body weight, rH(2)O was 28% lower in girls vs. boys (46 ± 7 vs. 64 ± 9 g·kg(-1)·day(-1)). Water input from food and beverages provided and metabolic production were 44 and 28% lower, respectively, in girls vs. boys. Urine and insensible water losses were 21 and 17% lower in girls vs. boys. BMI was positively associated with water turnover in both sexes (girls P = 0.037; boys P = 0.014). The intraclass correlation of rH(2)O between trials was 0.981 (P < 0.001). In conclusion, these overweight adolescents consumed water well in excess of sex-specific AI standards. The lower rH(2)O in girls compared to boys is consistent with adult females and males.

  1. Employee Turnover and Post Decision Accommodation Processes.

    DTIC Science & Technology

    1979-11-01

    1977; Dansereau et al., 1974; Koch and Steers, 1978, Waters et al., 1976; Krackhardt, McKenna , Porter and Steers, 1978). Variables such as these, when...worker. New York: Wiley, 1965. Krackhardt, D., McKenna , J., Porter, L. 14., and Steers, R. M. Coal- setting, supervisory behavior, and employee...consequences of turnover and to Larry Cummings, Daniel Ilgen, Terence R. Mitchell, Charles O’Reilly, and Barry Staw for their insightful and useful comments on

  2. Mechanosensitive activation of K+ channel via phospholipase C-induced depletion of phosphatidylinositol 4,5-bisphosphate in B lymphocytes.

    PubMed

    Nam, Joo Hyun; Lee, Hoo-Se; Nguyen, Yen Hoang; Kang, Tong Mook; Lee, Sung Won; Kim, Hye-Young; Kim, Sang Jeong; Earm, Yung E; Kim, Sung Joon

    2007-08-01

    In various types of cells mechanical stimulation of the plasma membrane activates phospholipase C (PLC). However, the regulation of ion channels via mechanosensitive degradation of phosphatidylinositol 4,5-bisphosphate (PIP(2)) is not known yet. The mouse B cells express large conductance background K(+) channels (LK(bg)) that are inhibited by PIP(2). In inside-out patch clamp studies, the application of MgATP (1 mm) also inhibited LK(bg) due to the generation of PIP(2) by phosphoinositide (PI)-kinases. In the presence of MgATP, membrane stretch induced by negative pipette pressure activated LK(bg), which was antagonized by PIP(2) (> 1 microm) or higher concentration of MgATP (5 mm). The inhibition by PIP(2) was partially reversible. However, the application of methyl-beta-cyclodextrin, a cholesterol scavenger disrupting lipid rafts, induced the full recovery of LK(bg) activity and facilitated the activation by stretch. In cell-attached patches, LK(bg) were activated by hypotonic swelling of B cells as well as by negative pressure. The mechano-activation of LK(bg) was blocked by U73122, a PLC inhibitor. Neither actin depolymerization nor the inhibition of lipid phosphatase blocked the mechanical effects. Direct stimulation of PLC by m-3M3FBS or by cross-linking IgM-type B cell receptors activated LK(bg). Western blot analysis and confocal microscopy showed that the hypotonic swelling of WEHI-231 induces tyrosine phosphorylation of PLCgamma2 and PIP(2) hydrolysis of plasma membrane. The time dependence of PIP(2) hydrolysis and LK(bg) activation were similar. The presence of LK(bg) and their stretch sensitivity were also proven in fresh isolated mice splenic B cells. From the above results, we propose a novel mechanism of stretch-dependent ion channel activation, namely, that the degradation of PIP(2) caused by stretch-activated PLC releases LK(bg) from the tonic inhibition by PIP(2).

  3. Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol 3-Kinase Signal Transduction by Altering Plasma Membrane Packing.

    PubMed

    Van Aelst, Britt; Devloo, Rosalie; Zachée, Pierre; t'Kindt, Ruben; Sandra, Koen; Vandekerckhove, Philippe; Compernolle, Veerle; Feys, Hendrik B

    2016-11-18

    Psoralen and ultraviolet A light (PUVA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft-versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC50), 8.4 ± 1.1 versus 4.3 ± 1.1 μm) and glycoprotein VI (EC50, 1.61 ± 0.85 versus 0.26 ± 0.21 μg/ml) but not PAR4 (EC50, 50 ± 1 versus 58 ± 1 μm) signal transduction. Our findings were confirmed in T-cells from graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases.

  4. Biochemical characterization of the tomato phosphatidylinositol-specific phospholipase C (PI-PLC) family and its role in plant immunity.

    PubMed

    Abd-El-Haliem, Ahmed M; Vossen, Jack H; van Zeijl, Arjan; Dezhsetan, Sara; Testerink, Christa; Seidl, Michael F; Beck, Martina; Strutt, James; Robatzek, Silke; Joosten, Matthieu H A J

    2016-09-01

    Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

  5. Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine.

    PubMed

    Preuss, Stefanie; Omam, Friede D; Scheiermann, Julia; Stadelmann, Sabrina; Winoto-Morbach, Supandi; von Bismarck, Philipp; Adam-Klages, Sabine; Knerlich-Lukoschus, Friederike; Lex, Dennis; Wesch, Daniela; Held-Feindt, Janka; Uhlig, Stefan; Schütze, Stefan; Krause, Martin F

    2012-11-01

    Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-β1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant "fortified" by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.

  6. A serum factor that activates the phosphatidylinositol phosphate signaling system in Xenopus oocytes.

    PubMed Central

    Tigyi, G; Dyer, D; Matute, C; Miledi, R

    1990-01-01

    Blood sera from many vertebrate species elicit large oscillatory chloride currents in oocytes from the frog Xenopus laevis. Rabbit serum was active at dilutions as great as one part in 10 million. Intracellularly applied serum was ineffective, and externally applied serum failed to trigger oscillatory currents when the intracellular level of ionized calcium was prevented from rising by loading the oocyte with EGTA. The serum also caused an increase of inositol 1,4,5-trisphosphate in the oocyte. We conclude that serum contains a factor which activates a membrane receptor that is coupled to the phosphatidylinositol second messenger system. The active factor is a protein with an apparent molecular mass of 60-70 kDa in gel permeation chromatography. Although the normal function of the serum factor is still unknown, it may have far-reaching implications, because it acts on the multifunctional phosphatidylinositol phosphate signaling system. Also, because of its great potency the serum factor and Xenopus oocytes are very useful for probing the operation of the phosphatidylinositol system. PMID:1689488

  7. Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean.

    PubMed

    Harryson, P.; Morre, D. J.; Sandelius, A. S.

    1996-02-01

    Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity.

  8. Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean.

    PubMed Central

    Harryson, P.; Morre, D. J.; Sandelius, A. S.

    1996-01-01

    Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity. PMID:12226209

  9. Syntaxin4 Interacting Protein (Synip) Binds Phosphatidylinositol (3,4,5) Triphosphate

    PubMed Central

    Saito, Tsugumichi; Okada, Shuichi; Nohara, Atsushi; Tagaya, Yuko; Osaki, Aya; Oh-i, Shinsuke; Takahashi, Hiroki; Tsuchiya, Takafumi; Hashimoto, Koshi; Satoh, Tetsurou; Yamada, Masanobu; Pessin, Jeffrey E.; Mori, Masatomo

    2012-01-01

    The insulin responsive Glut4 transport vesicles contain the v-SNARE protein Vamp2 that associate with the plasma membrane t-SNARE protein Syntaxin 4 to drive insulin-stimulated Glut4 translocation in skeletal muscle and adipocytes. The syntaxin 4 interacting protein (Synip) binds to syntaxin 4 in the basal state and dissociates in the insulin-stimulated state allowing for the subsequent binding of Vamp2 containing Glut4 vesicles and fusion with the plasma membrane. In this study, we have found that Synip binds phosphatidylinositol 3,4,5-triphosphate (PIP3), but not phosphatidylinositol 3 phosphate (PIP) or phosphatidylinositol 3,4-biphosphate (PIP2) through the Synip WW domain as deletion of this domain (Synip ΔWW) failed to bind PIP3. Over-expressed Synip ΔWW in 3T3L1 adipocytes reduced the basal levels of Glut4 at the plasma membrane with no effect on the binding to syntaxin 4 in vitro. Subcellular fractionation demonstrated that the amount of Synip ΔWW at the PM was decreased in response to insulin in 3T3L1 adipocytes whereas the amount of Synip WT increased. These data suggest that in the presence of insulin, the dissociated Synip remains anchored to the plasma membrane by binding to PIP3. PMID:22880106

  10. Phosphatidylinositol 4,5-bisphosphate is required for invasive growth in Saccharomyces cerevisiae.

    PubMed

    Guillas, Isabelle; Vernay, Aurélia; Vitagliano, Jean-Jacques; Arkowitz, Robert A

    2013-08-15

    Phosphatidylinositol phosphates are important regulators of processes such as the cytoskeleton organization, membrane trafficking and gene transcription, which are all crucial for polarized cell growth. In particular, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has essential roles in polarized growth as well as in cellular responses to stress. In the yeast Saccharomyces cerevisiae, the sole phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) Mss4p is essential for generating plasma membrane PtdIns(4,5)P2. Here, we show that Mss4p is required for yeast invasive growth in low-nutrient conditions. We isolated specific mss4 mutants that were defective in cell elongation, induction of the Flo11p flocculin, adhesion and cell wall integrity. We show that mss4-f12 cells have reduced plasma membrane PtdIns(4,5)P2 levels as well as a defect in its polarized distribution, yet Mss4-f12p is catalytically active in vitro. In addition, the Mss4-f12 protein was defective in localizing to the plasma membrane. Furthermore, addition of cAMP, but not an activated MAPKKK allele, partially restored the invasive growth defect of mss4-f12 cells. Taken together, our results indicate that plasma membrane PtdIns(4,5)P2 is crucial for yeast invasive growth and suggest that this phospholipid functions upstream of the cAMP-dependent protein kinase A signaling pathway.

  11. Inositol lipids: from an archaeal origin to phosphatidylinositol 3,5-bisphosphate faults in human disease.

    PubMed

    Michell, Robert H

    2013-12-01

    The last couple of decades have seen an extraordinary transformation in our knowledge and understanding of the multifarious biological roles of inositol phospholipids. Herein, I briefly consider two topics. The first is the role that recently acquired biochemical and genomic information - especially from archaeons - has played in illuminating the possible evolutionary origins of the biological employment of inositol in lipids, and some questions that these studies raise about the 'classical' biosynthetic route to phosphatidylinositol. The second is the growing recognition of the importance in eukaryotic cells of phosphatidylinositol 3,5-bisphosphate. Phosphatidylinositol 3,5-bisphosphate only entered our phosphoinositide consciousness quite recently, but it is speedily gathering a plethora of roles in diverse cellular processes and diseases thereof. These include: control of endolysosomal vesicular trafficking and of the activity of ion channels and pumps in the endolysosomal compartment; control of constitutive and stimulated protein traffic to and from plasma membrane subdomains; control of the nutrient and stress-sensing target of rapamycin complex 1 pathway (TORC1); and regulation of key genes in some central metabolic pathways.

  12. Generational differences in registered nurse turnover.

    PubMed

    LeVasseur, Sandra A; Wang, Chen-Yen; Mathews, Barbara; Boland, Mary

    2009-08-01

    The chronic nature of the nursing workforce shortage in the United States is a continuing concern. As the nationwide gap between supply and demand grows, it remains unknown what impact turnover will have on nursing, access to care, and efforts to improve quality and safety of health care. It also remains unclear whether the recent turnover trends among new graduate registered nurses differ from past generational cohorts of new nurses. The aims of this study were to identify the reasons why registered nurses turnover by generational cohort (Veterans, Baby Boomers, and GenXMs) and to compare the length of time nurses were employed in their first five nursing positions by generational cohort. The findings suggest the three generational cohorts displayed similar reasons for leaving nursing positions with relocation, career advancement, and personal/family reasons reported most frequently. Except for the first nursing position, significant generational effects were found in the length of time Veterans, Baby Boomer, and GenXMs stayed employed in their nursing positions. It remains unknown why the GenXMs displayed a significantly shorter length of employment time in their second, third, fourth, and fifth nursing positions. The decline in length of employment time displayed in both the Baby Boomers and GenXMs may be an issue of concern requiring future research.

  13. Glucose turnover and recycling in colorectal carcinoma.

    PubMed

    Kokal, W A; McCulloch, A; Wright, P D; Johnston, I D

    1983-11-01

    Glucose metabolism is affected by various pathologic states including tumors. In this project, glucose turnover and recycling rates in 11 patients with colorectal carcinoma were measured using a double-labelled 3-3H and 1-14C glucose injection technique. Fasting blood glucose, lactate, pyruvate, alanine, glycerol, 3-hydroxybutyrate, acetoacetate, plasma cortisol, and plasma insulin concentrations were also measured. No patient in the study had a history of diabetes mellitus or endocrine disorders, nor any abnormal liver function tests. The findings demonstrated a significantly elevated glucose turnover rate in patients with Dukes C and D lesions in comparison to patients with Dukes B lesions. Cori recycling rates were not significantly different between Dukes B vs. Dukes C and D patients. There were no differences between Dukes B and Dukes C and D patients in any of the metabolites measured. Furthermore, there were no significant differences in glucose turnover or recycling rates as a function of pre-illness weight loss. These data suggest that, when colorectal carcinoma extends beyond the limits of the bowel wall, glucose metabolism is significantly altered.

  14. Quantitative analysis of protein turnover in plants.

    PubMed

    Nelson, Clark J; Li, Lei; Millar, A Harvey

    2014-03-01

    Proteins are constantly being synthesised and degraded as plant cells age and as plants grow, develop and adapt the proteome. Given that plants develop through a series of events from germination to fruiting and even undertake whole organ senescence, an understanding of protein turnover as a fundamental part of this process in plants is essential. Both synthesis and degradation processes are spatially separated in a cell across its compartmented structure. The majority of protein synthesis occurs in the cytosol, while synthesis of specific components occurs inside plastids and mitochondria. Degradation of proteins occurs in both the cytosol, through the action of the plant proteasome, and in organelles and lytic structures through different protease classes. Tracking the specific synthesis and degradation rate of individual proteins can be undertaken using stable isotope feeding and the ability of peptide MS to track labelled peptide fractions over time. Mathematical modelling can be used to follow the isotope signature of newly synthesised protein as it accumulates and natural abundance proteins as they are lost through degradation. Different technical and biological constraints govern the potential for the use of (13)C, (15)N, (2)H and (18)O for these experiments in complete labelling and partial labelling strategies. Future development of quantitative protein turnover analysis will involve analysis of protein populations in complexes and subcellular compartments, assessing the effect of PTMs and integrating turnover studies into wider system biology study of plants. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Forest turnover rates follow global and regional patterns of productivity

    USGS Publications Warehouse

    Stephenson, N.L.; van Mantgem, P.J.

    2005-01-01

    Using a global database, we found that forest turnover rates (the average of tree mortality and recruitment rates) parallel broad-scale patterns of net primary productivity. First, forest turnover was higher in tropical than in temperate forests. Second, as recently demonstrated by others, Amazonian forest turnover was higher on fertile than infertile soils. Third, within temperate latitudes, turnover was highest in angiosperm forests, intermediate in mixed forests, and lowest in gymnosperm forests. Finally, within a single forest physiognomic type, turnover declined sharply with elevation (hence with temperature). These patterns of turnover in populations of trees are broadly similar to the patterns of turnover in populations of plant organs (leaves and roots) found in other studies. Our findings suggest a link between forest mass balance and the population dynamics of trees, and have implications for understanding and predicting the effects of environmental changes on forest structure and terrestrial carbon dynamics. ??2005 Blackwell Publishing Ltd/CNRS.

  16. Norepinephrine turnover in brown adipose tissue is stimulated by a single meal

    SciTech Connect

    Glick, Z.; Raum, W.J.

    1986-07-01

    A single meal stimulates brown adipose tissue (BAT) thermogenesis in rats. In the present study the role of norepinephrine in this thermogenic response was assessed from the rate of its turnover in BAT after a single test meal. For comparison, norepinephrine turnover was determined in the heart and spleen. A total of 48 male Wistar rats (200 g) were trained to eat during two feeding sessions per day. On the experimental day, one group (n = 24) was meal deprived and the other (n = 24) was given a low-protein high-carbohydrate test meal for 2 h. The synthesis inhibition method with ..cap alpha..-methyl-p-tyrosine was employed to determine norepinephrine turnover from its concentration at four hourly time points after the meal. Tissue concentrations of norepinephrine were determined by radioimmunoassay. Norepinephrine concentration and turnover rate were increased more than threefold in BAT of the meal-fed compared with the meal-deprived rats. Neither were significantly altered by the meal in the heart or spleen. The data suggest that norepinephrine mediates a portion of the thermic effect of meals that originate in BAT.

  17. Inhibitory effect of capsaicin on B16-F10 melanoma cell migration via the phosphatidylinositol 3-kinase/Akt/Rac1 signal pathway

    PubMed Central

    Shin, Dong-Hoon; Kim, Ok-Hee; Jun, Hye-Seung

    2008-01-01

    Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient of red pepper, has been reported to possess anti-carcinogenic and anti-mutagenic activities. In this study, the anti-migration activity of capsaicin on highly metastatic B16-F10 melanoma cells was investigated. Capsaicin significantly inhibited the migration of melanoma cells without showing obvious cellular cytotoxicity at low doses. This effect correlated with the down-regulation of phosphatidylinositol 3-kinase (PI3-K) and its downstream target, Akt. Although B16-F10 cell migration was increased by the PI3-K activator through the activation of Akt, these PI3-K activator-induced phenomena were attenuated by capsaicin. Moreover, capsaicin was found to significantly inhibit Rac1 activity in a pull-down assay. These results demonstrate that capsaicin inhibits the migration of B16-F10 cells through the inhibition of the PI3-K/Akt/Rac1 signal pathway. The present investigation suggests that capsaicin targets PI3-K/Akt/Rac1-mediated cellular events in B16-F10 melanoma cells. Consequently, capsaicin administration should be considered an effective approach for the suppression of invasion and metastasis in malignant melanoma chemotherapy. PMID:18985006

  18. Treatment staff turnover in organizations implementing evidence-based practices: turnover rates and their association with client outcomes.

    PubMed

    Garner, Bryan R; Hunter, Brooke D; Modisette, Kathryn C; Ihnes, Pamela C; Godley, Susan H

    2012-03-01

    High staff turnover has been described as a problem for the substance use disorder treatment field. This assertion is based primarily on the assumption that staff turnover adversely impacts treatment delivery and effectiveness. This assumption, however, has not been empirically tested. In this study, we computed annualized rates of turnover for treatment staff (N = 249) participating in an evidence-based practice implementation initiative and examined the association between organizational-level rates of staff turnover and client-level outcomes. Annualized rates of staff turnover were 31% for clinicians and 19% for clinical supervisors. In addition, multilevel analyses did not reveal the expected relationship between staff turnover and poorer client-level outcomes. Rather, organizational-level rates of staff turnover were found to have a significant positive association with two measures of treatment effectiveness: less involvement in illegal activity and lower social risk. Possible explanations for these findings are discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Treatment staff turnover in organizations implementing evidence-based practices: Turnover rates and their association with client outcomes

    PubMed Central

    Garner, Bryan R.; Hunter, Brooke D.; Modisette, Kathryn C.; Ihnes, Pamela C.; Godley, Susan H.

    2011-01-01

    High staff turnover has been described as a problem for the substance use disorder treatment field. This assertion is based primarily on the assumption that staff turnover adversely impacts treatment delivery and effectiveness. This assumption, however, has not been empirically tested. In this study, we computed annualized rates of turnover for treatment staff (n=249) participating in an evidence-based practice implementation initiative and examined the association between organizational-level rates of staff turnover and client-level outcomes. Annualized rates of staff turnover were 31% for clinicians and 19% for clinical supervisors. Additionally, multilevel analyses did not reveal the expected relationship between staff turnover and poorer client-level outcomes. Rather, organizational-level rates of staff turnover were found to have a significant positive association with two measures of treatment effectiveness: less involvement in illegal activity and lower social risk. Possible explanations for these findings are discussed. PMID:22154040

  20. Turnover of adenosine in plasma of human and dog blood

    SciTech Connect

    Moeser, G.H.S.; Schrader, J.; Deussen, A.

    1989-04-01

    To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a ''stopping solution.'' Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added (/sup 3/H)adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not correlate well with changes in blood flow but were augmented in the presence of dipyridamole.

  1. Dynamic aspects of voluntary turnover: an integrated approach to curvilinearity in the performance-turnover relationship.

    PubMed

    Becker, William J; Cropanzano, Russell

    2011-03-01

    Previous research pertaining to job performance and voluntary turnover has been guided by 2 distinct theoretical perspectives. First, the push-pull model proposes that there is a quadratic or curvilinear relationship existing between these 2 variables. Second, the unfolding model of turnover posits that turnover is a dynamic process and that a downward performance change may increase the likelihood of organizational separation. Drawing on decision theory, we propose and test an integrative framework. This approach incorporates both of these earlier models. Specifically, we argue that individuals are most likely to voluntarily exit when they are below-average performers who are also experiencing a downward performance change. Furthermore, the interaction between this downward change and performance partially accounts for the curvilinear relationship proposed by the push-pull model. Findings from a longitudinal field study supported this integrative theory.

  2. Turnover in health care: the mediating effects of employee engagement.

    PubMed

    Collini, Stevie A; Guidroz, Ashley M; Perez, Lisa M

    2015-03-01

    This study aimed to understand the interaction between interpersonal respect, diversity climate, mission fulfilment and engagement to better predict turnover in health care. Registered nurse turnover has averaged 14% and current nursing shortages are expected to spread. Few studies have studied employee engagement as a mediator between organisational context and turnover. Study participants were employees working within 185 departments across ten hospitals within a large healthcare organisation in the USA. Although a total of 5443 employees work in these departments, employee opinion survey responses were aggregated by department before being linked to turnover rates gathered from company records. Engagement fully mediated the relationship between respect and turnover and the relationship between mission fulfilment and turnover. Diversity climate was not related to turnover. Turnover in health care poses a significant threat to the mission of creating a healing environment for patients and these results demonstrate that workplace respect and connection to the mission affect turnover by decreasing engagement. The findings demonstrated that to increase engagement, and improve turnover rates in health care, it would be beneficial for organisations, and nurse management to focus on improving mission fulfilment and interpersonal relationships. © 2013 John Wiley & Sons Ltd.

  3. Turnover of xanthoma cholesterol in hyperlipoproteinemia patients.

    PubMed

    Bhattacharyya, A K; Connor, W E; Mausolf, F A; Flatt, A D

    1976-03-01

    The turnover of xanthoma cholesterol was measured in 9 hyperlipidemic and one normocholesterolemic patients. Sequential biopsies of the xanthomas were obtained 13 to 364 days after the administration of isotopic cholesterol and were then analyzed for cholesterol specific activity. A total of 34 xanthomas of 3 different types - 10 tendon xanthomas, 3 tuberous xanthomas, and 21 xanthelasmas - comprised the material for analysis. The cholesterol specific activity ratio of tendron xanthomas to that of the plasma varied from 11 per cent at 21 days to a maximum of 543 per cent at 122 days after the intravenous administration of isotopic cholesterol. This ratio declined to 426 per cent at 182 days and was still 131 per cent at 364 days. Similarly, the cholesterol specific activity of xanthelasmas increased gradually. In most instances, the xanthelasma cholesterol attained isotopic equilibration with plasma cholesterol by about 50 days but varied from patient to patient (minimum time, 46 days and maximum time, 91 days). The cholesterol content of xanthomas ranged from 10.7 to 197.0 mg per gram of dry weight of the tissue. Sixty-one to 87 per cent of the total xanthoma cholesterol was esterified. No other sterols were identified in these xanthomas. Thus, the cholesterol of 3 types of xanthoma readily attained isotopic equilibration with the plasma cholesterol which suggested total exchangeability of cholesterol between plasma and xanthomas. The uptake of cholesterol by the xanthomas from plasma was rapid considering the large mass of cholesterol in the lesions. The turnover of xanthoma cholesterol was intermediate between that of the rapidly exchangeable pool and of the slowly exchangeable pool of body cholesterol. Comparison of these results with those obtained in human advanced atheroma suggest that the turnover of xanthoma cholesterol and atheroma cholesterol are quite different.

  4. Platelet Turnover Predicts Outcome after Coronary Intervention

    PubMed Central

    Iliev, Liana; Bruno, Veronika; Rohla, Miklos; Egger, Florian; Weiss, Thomas W.; Hübl, Wolfgang; Willheim, Martin; Wojta, Johann; Huber, Kurt

    2017-01-01

    Summary Elevated platelet turnover contributes to high platelet reactivity. High platelet reactivity after percutaneous coronary intervention (PCI) is associated with major adverse cardiovascular events (MACE). The purpose of this study was to determine the prognostic value of platelet turnover and function with regard to MACE after PCI with stent implantation. In this prospective observational study, 486 consecutive patients after PCI on aspirin and clopidogrel were included to determine platelet turnover (mean platelet volume (MPV), reticulated platelet fraction (RPF)) and platelet function (multiple electrode aggregometry (MEA), vasodilator-stimulated phosphoprotein-phosphorylation (VASP-P) assay). At six-months follow-up, MACE occurred in 10.7 % of patients. RPF (odds ratio [OR]=1.173 (95% confidence interval [CI 95 %] 1.040–1.324), p=0.009) and MPV (OR=1.459 (CI 95 % 1.059–2.008), p=0.021) were univariable predictors of MACE, whereas VASP-P (OR=1.016 (CI 95 % 1.000–1.032), p=0.052) and MEA (OR=0.999 (CI 95 % 0.980–1.017), p=0.895) failed to predict MACE. RPF remained the only platelet variable independently associated with MACE. The best model to predict MACE included: troponin I (OR=1.007 (CI 95 % 1.002–1.012), p=0.009), RPF (OR=1.136 (CI 95 % 1.001–1.288), p=0.048), CRP (OR=1.008 (CI 95 % 1.001–1.014), p=0.023) and history of myocardial infarction (OR=2.039 (CI 95 % 1.093–3.806), p=0.025). RPF (OR=1.211 (CI 95 % 1.042–1.406), p=0.012) was also independently associated with in-hospital bleedings. In conclusion, RPF as index of platelet turnover is an independent predictor of MACE and bleeding events in PCI patients on dual antiplatelet therapy. Since RPF can reliably be quantified along with routine haemograms, RPF might easily be applied in the setting of cardiovascular risk prediction. PMID:28229159

  5. Organizational commitment and turnover of nursing home administrators.

    PubMed

    Castle, Nicholas G

    2006-01-01

    In this investigation, the associations between organizational commitment (OC), intent-to-turnover, and actual turnover of a large sample of nursing home administrators (NHAs) are examined. Data used come from a mail survey, from which 632 responses were received from the NHAs (response rate = 63%). The one-year turnover rate of NHAs was 39 percent, and in almost all cases (87%) these NHAs had also exhibited low OC scores. The intent-to-turnover results show thinking about quitting comes before searching for a new position, which in turn both comes before the intention to quit. Multivariate analyses show work overload has a strong and robust association with both intent-to-turnover and turnover of NHAs, and may indicate that NHAs are leaving their positions because they are understaffed.

  6. Mentor program boosts new nurses' satisfaction and lowers turnover rate.

    PubMed

    Fox, Kathy C

    2010-07-01

    In 2004, the turnover rate among first-year registered nurses (RNs) at St. Francis Hospital and Health Centers had mushroomed to 31%. Based on research, in 2006, the hospital embarked on a journey to implement an RN mentor program to improve satisfaction and reduce turnover. A pilot program was initiated, including 12 RN mentors and 12 RN protégés from select nursing units. The results showed a 0% turnover rate during the 1-year pilot program. Based on these findings, the mentor program was expanded to include RNs working in inpatient nursing units and surgery and emergency departments. Each year, the RN turnover rate has decreased. In 2009, the turnover rate was 10.3%. Because of the success of the program, it has been expanded in scope to include other professionals experiencing high turnover in targeted departments, including radiological technicians, respiratory therapists, pharmacists, and physical therapists.

  7. Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-Kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity

    SciTech Connect

    Xie,P.; Williams, D.; Atilla-Gokcumen, G.; Milk, L.; Xiao, M.; Smalley, K.; Herlyn, M.; Meggers, E.; Marmorstein, R.

    2008-01-01

    Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3K?/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.

  8. Collagen and tissue turnover as a function of age: Implications for fibrosis.

    PubMed

    Karsdal, Morten A; Genovese, Federica; Madsen, Emilie A; Manon-Jensen, Tina; Schuppan, Detlef

    2016-01-01

    The extracellular matrix (ECM) is the backbone of all tissues. It is a complex grid consisting of multiple structural proteins which each play a vital role for the function and maintenance of normal tissue function. In development and growth, tissue is being formed and elaborated (tissue modeling), while in adult life, tissues are being maintained and remodeled. These processes involve likely different mechanisms. During tissue modeling and remodeling, small fragments of proteins are released into the circulation, where they may be used as biomarkers for tissue turnover. The aim of the study was to investigate ECM turnover in rodents as a function of age. Serum of rats of 1, 2, 3, 4, 5, 6, 10 and 12months of age was profiled for 15 markers of ECM turnover, including: fragments of type I, II, III, IV, V and VI collagen formation (P1NP, P4NP-7S, Pro-C5, Pro-C6) and degradation (C1M, C2M, C2M-beta, C3M, C4M, C5M, C6M); biglycan (BGM) and elastin (ELM7) degradation; and the type I and II collagen telopeptides CTX-I and CTX-II. Type I and II collagen turnover was up to 93% and 97% downregulated in old (one year) compared to young (one month) old animals (p<0.0001), while type IV and V collagen and biglycan turnover was upregulated 2.5-, 2- and 2-fold, respectively (p<0.0001). Type III and VI collagen and elastin turnover was not influenced significantly by age. ECM turnover rates were consistently different in young vs. old animals, up to 30 fold. This appears to be due to body growth, a different ECM composition and a higher regenerative capability of connective tissues in young vs. old animals. These changes have to be accounted for in translational science. Both in measuring serum levels of ECM biomarkers and in the development of therapies to speed up wound healing or inhibit fibrogenesis. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  9. Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

    PubMed

    Fujimoto, Masaru; Suda, Yasuyuki; Vernhettes, Samantha; Nakano, Akihiko; Ueda, Takashi

    2015-02-01

    The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.

  10. Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization.

    PubMed

    Zhuang, Zhi-Ye; Xu, Haoxing; Clapham, David E; Ji, Ru-Rong

    2004-09-22

    Although the PI3K (phosphatidylinositol 3-kinase) pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in neural plasticity. It is unknown whether the PI3K pathway can mediate pain hypersensitivity. Intradermal injection of capsaicin and NGF produce heat hyperalgesia by activating their respective TRPV1 (transient receptor potential vanilloid receptor-1) and TrkA receptors on nociceptor sensory nerve terminals. We examined the activation of PI3K in primary sensory DRG neurons by these inflammatory agents and the contribution of PI3K activation to inflammatory pain. We further investigated the correlation between the PI3K and the ERK (extracellular signal-regulated protein kinase) pathway. Capsaicin and NGF induce phosphorylation of the PI3K downstream target AKT (protein kinase B), which is blocked by the PI3K inhibitors LY294002 and wortmannin, indicative of the activation of PI3K by both agents. ERK activation by capsaicin and NGF was also blocked by PI3K inhibitors. Similarly, intradermal capsaicin in rats activated PI3K and ERK in C-fiber DRG neurons and epidermal nerve fibers. Injection of PI3K or MEK (ERK kinase) inhibitors into the hindpaw attenuated capsaicin- and NGF-evoked heat hyperalgesia but did not change basal heat sensitivity. Furthermore, PI3K, but not ERK, inhibition blocked early induction of hyperalgesia. In acutely dissociated DRG neurons, the capsaicin-induced TRPV1 current was strikingly potentiated by NGF, and this potentiation was completely blocked by PI3K inhibitors and primarily suppressed by MEK inhibitors. Therefore, PI3K induces heat hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent manner. The PI3K pathway also appears to play a role that is distinct from ERK by regulating the early onset of inflammatory pain.

  11. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

    PubMed

    Campbell, Paul M; Groehler, Angela L; Lee, Kwang M; Ouellette, Michel M; Khazak, Vladimir; Der, Channing J

    2007-03-01

    Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

  12. Klebsiella pneumoniae Translocates across the Intestinal Epithelium via Rho GTPase- and Phosphatidylinositol 3-Kinase/Akt-Dependent Cell Invasion

    PubMed Central

    Hsu, Chun-Ru; Pan, Yi-Jiun; Liu, Ju-Yun; Chen, Chun-Tang; Lin, Tzu-Lung

    2014-01-01

    Klebsiella pneumoniae is an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests that K. pneumoniae infections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine how K. pneumoniae penetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization of K. pneumoniae in three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that these K. pneumoniae isolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore, K. pneumoniae appeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important for K. pneumoniae invasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminished K. pneumoniae invasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization, in vivo invasion of K. pneumoniae into colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation for K. pneumoniae. Cell invasion by manipulating host machinery provides a pathway for gut-colonized K. pneumoniae cells to penetrate the intestinal barrier and access extraintestinal locations to cause disease. PMID:25452552

  13. Klebsiella pneumoniae translocates across the intestinal epithelium via Rho GTPase- and phosphatidylinositol 3-kinase/Akt-dependent cell invasion.

    PubMed

    Hsu, Chun-Ru; Pan, Yi-Jiun; Liu, Ju-Yun; Chen, Chun-Tang; Lin, Tzu-Lung; Wang, Jin-Town

    2015-02-01

    Klebsiella pneumoniae is an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests that K. pneumoniae infections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine how K. pneumoniae penetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization of K. pneumoniae in three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that these K. pneumoniae isolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore, K. pneumoniae appeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important for K. pneumoniae invasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminished K. pneumoniae invasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization, in vivo invasion of K. pneumoniae into colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation for K. pneumoniae. Cell invasion by manipulating host machinery provides a pathway for gut-colonized K. pneumoniae cells to penetrate the intestinal barrier and access extraintestinal locations to cause disease. Copyright © 2015, American Society for Microbiology. All

  14. Reverse signaling via a glycosyl-phosphatidylinositol-linked ephrin prevents midline crossing by migratory neurons during embryonic development in Manduca.

    PubMed

    Coate, Thomas M; Wirz, Jacqueline A; Copenhaver, Philip F

    2008-04-09

    We have investigated whether reverse signaling via a glycosyl-phosphatidylinositol (GPI)-linked ephrin controls the behavior of migratory neurons in vivo. During the formation of the enteric nervous system (ENS) in the moth Manduca, approximately 300 neurons [enteric plexus (EP) cells] migrate onto the midgut via bilaterally paired muscle bands but avoid adjacent midline regions. As they migrate, the EP cells express a single ephrin ligand (MsEphrin; a GPI-linked ligand), whereas the midline cells express the corresponding Eph receptor (MsEph). Blocking endogenous MsEphrin-MsEph receptor interactions in cultured embryos resulted in aberrant midline crossing by the neurons and their processes. In contrast, activating endogenous MsEphrin on the EP cells with dimeric MsEph-Fc constructs inhibited their migration and outgrowth, supporting a role for MsEphrin-dependent reverse signaling in this system. In short-term cultures, blocking endogenous MsEph receptors allowed filopodia from the growth cones of the neurons to invade the midline, whereas activating neuronal MsEphrin led to filopodial retraction. MsEphrin-dependent signaling may therefore guide the migratory enteric neurons by restricting the orientation of their leading processes. Knocking down MsEphrin expression in the EP cells with morpholino antisense oligonucleotides also induced aberrant midline crossing, consistent with the effects of blocking endogenous MsEphrin-MsEph interactions. Unexpectedly, this treatment enhanced the overall extent of migration, indicating that MsEphrin-dependent signaling may also modulate the general motility of the EP cells. These results demonstrate that MsEphrin-MsEph receptor interactions normally prevent midline crossing by migratory neurons within the developing ENS, an effect that is most likely mediated by reverse signaling through this GPI-linked ephrin ligand.

  15. Phosphatidylinositol-3-kinase pathway aberrations in gastric and colorectal cancer: meta-analysis, co-occurrence and ethnic variation.

    PubMed

    Chong, Mei-Ling; Loh, Marie; Thakkar, Bhavin; Pang, Brendan; Iacopetta, Barry; Soong, Richie

    2014-03-01

    Inhibition of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a cancer treatment strategy that has entered into clinical trials. We performed a meta-analysis on the frequency of prominent genetic (PIK3CA mutation, PIK3CA amplification and PTEN deletion) and protein expression (high PI3K, PTEN loss and high pAkt) aberrations in the PI3K pathway in gastric cancer (GC) and colorectal cancer (CRC). We also performed laboratory analysis to investigate the co-occurrence of these aberrations. The meta-analysis indicated that East Asian and Caucasian GC patients differ significantly for the frequencies of PIK3CA Exon 9 and 20 mutations (7% vs. 15%, respectively), PTEN deletion (21% vs. 4%) and PTEN loss (47% vs. 78%), while CRC patients differed for PTEN loss (57% vs. 26%). High study heterogeneity (I(2) > 80) was observed for all aberrations except PIK3CA mutations. Laboratory analysis of tumors from East Asian patients revealed significant differences between GC (n = 79) and CRC (n = 116) for the frequencies of PIK3CA amplification (46% vs. 4%) and PTEN loss (54% vs. 78%). The incidence of GC cases with 0, 1, 2 and 3 concurrent aberrations was 14%, 52%, 27% and 8%, respectively, while for CRC it was 10%, 60%, 25% and 4%, respectively. Our study consolidates knowledge on the frequency, co-occurrence and clinical relevance of PI3K pathway aberrations in GC and CRC. Up to 86% of GC and 90% of CRC have at least one aberration in the PI3K pathway, and there are significant differences in the frequencies of these aberrations according to cancer type and ethnicity.

  16. Phosphatidylinositol 3-Kinase Mediates β-Catenin Dysfunction of Airway Epithelium in a Toluene Diisocyanate-Induced Murine Asthma Model.

    PubMed

    Yao, Lihong; Zhao, Haijin; Tang, Haixiong; Song, Jiafu; Dong, Hangming; Zou, Fei; Cai, Shaoxi

    2015-09-01

    Cell-cell junctions are critical for the maintenance of cellular as well as tissue polarity and integrity. Yet the role of phosphatidylinositol 3-kinase (PI3K) in dysregulation of airway epithelial adherens junctions in toluene diisocyanate (TDI)-induced asthma has not been addressed. Male BALB/c mice were first dermally sensitized and then challenged with TDI by means of compressed air nebulization. The mice were treated intratracheally with PI3K inhibitor LY294002. Levels of phospho-Akt in airway epithelium and whole lung tissues were markedly increased in TDI group compared with control mice, which decreased after administration of LY294002. The dilated intercellular spaces of airway epithelium induced by TDI were partially recovered by LY294002. Both the protein expression and distribution of adherens junction proteins E-cadherin and β-catenin were altered by TDI. Treatment with LY294002 rescued the distribution of E-cadherin and β-catenin at cell-cell membranes, restored total β-catenin pool, but had no effect on protein level of E-cadherin. At the same time, LY294002 also inhibited phosphorylation of ERK, glycogen synthase kinase3β and tyrosine 654 of β-catenin induced by TDI. In summary, our results showed that the PI3K pathway mediates β-catenin dysregulation in a TDI-induced murine asthma model, which may be associated with increased tyrosine phosphorylation of β-catenin. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Hexosamine biosynthesis pathway flux contributes to insulin resistance via altering membrane phosphatidylinositol 4,5-bisphosphate and cortical filamentous actin.

    PubMed

    Bhonagiri, Padma; Pattar, Guruprasad R; Horvath, Emily M; Habegger, Kirk M; McCarthy, Alicia M; Elmendorf, Jeffrey S

    2009-04-01

    We recently found that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2))-regulated filamentous actin (F-actin) polymerization was diminished in hyperinsulinemic cell culture models of insulin resistance. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes the PIP(2)/F-actin dysregulation and insulin resistance induced by hyperinsulinemia. Increased HBP activity was detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin for 12 h) and in cells where HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP(2) and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP(2) corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP(2)/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored PIP(2)-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of glutamine:fructose-6-phosphate amidotransferase was associated with a loss of detectable plasma membrane PIP(2) and insulin sensitivity. A slight decrease in intracellular ATP resulted from amplifying HBP by hyperinsulinemia and GlcN. However, experimental maintenance of the intracellular ATP pool under both conditions with inosine did not reverse the PIP(2)/F-actin-based insulin-resistant state. Furthermore, less invasive challenges with glucose, in the absence of insulin, also led to PIP(2)/F-actin dysregulation. Accordingly, we suggest that the functionality of cell systems dependent on PIP(2) and/or F-actin status, such as the glucose transport system, can be critically compromised by inappropriate HBP activity.

  18. Hexosamine Biosynthesis Pathway Flux Contributes to Insulin Resistance via Altering Membrane Phosphatidylinositol 4,5-Bisphosphate and Cortical Filamentous Actin

    PubMed Central

    Bhonagiri, Padma; Pattar, Guruprasad R.; Horvath, Emily M.; Habegger, Kirk M.; McCarthy, Alicia M.; Elmendorf, Jeffrey S.

    2009-01-01

    We recently found that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) polymerization was diminished in hyperinsulinemic cell culture models of insulin resistance. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes the PIP2/F-actin dysregulation and insulin resistance induced by hyperinsulinemia. Increased HBP activity was detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin for 12 h) and in cells where HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP2 and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP2 corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP2/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored PIP2-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of glutamine:fructose-6-phosphate amidotransferase was associated with a loss of detectable plasma membrane PIP2 and insulin sensitivity. A slight decrease in intracellular ATP resulted from amplifying HBP by hyperinsulinemia and GlcN. However, experimental maintenance of the intracellular ATP pool under both conditions with inosine did not reverse the PIP2/F-actin-based insulin-resistant state. Furthermore, less invasive challenges with glucose, in the absence of insulin, also led to PIP2/F-actin dysregulation. Accordingly, we suggest that the functionality of cell systems dependent on PIP2 and/or F-actin status, such as the glucose transport system, can be critically compromised by inappropriate HBP activity. PMID:19036880

  19. Randomized phase 1 study of the phosphatidylinositol 3-kinase δ inhibitor idelalisib in patients with allergic rhinitis.

    PubMed

    Horak, Friedrich; Puri, Kamal D; Steiner, Bart H; Holes, Leanne; Xing, Guan; Zieglmayer, Petra; Zieglmayer, René; Lemell, Patrick; Yu, Albert

    2016-06-01

    Phosphatidylinositol 3-kinase p110δ isoform (PI3K p110δ) activity is essential for mast cell activation, suggesting that inhibition of PI3K p110δ might be useful in treating allergic diseases. We sought to determine the effect of the PI3K p110δ-selective inhibitor idelalisib on allergic responses. This phase 1 randomized, double-blind, placebo-controlled, 2-period crossover study was conducted with the Vienna Challenge Chamber. Grass pollen-induced allergic symptoms were documented during screening. Eligible subjects received idelalisib (100 mg twice daily) or placebo for 7 days, with allergen challenge on day 7. After a 2-week washout period, subjects received the alternate treatment and repeated allergen challenge. Study measures included safety, nasal and nonnasal symptoms, nasal airflow, nasal secretions, basophil activation, and plasma cytokine levels. Forty-one patients with allergic rhinitis received idelalisib/placebo (n = 21) or placebo/idelalisib (n = 20). Idelalisib treatment was well tolerated. Mean total nasal symptom scores were lower during the combined idelalisib treatment periods compared with placebo (treatment difference [idelalisib - placebo], -1.78; 95% CI, -2.53 to -1.03; P < .001). Statistically significant differences were also observed for the combined treatment periods for total symptom scores, nasal airflow, nasal secretion weight, and nasal congestion scores. The percentage of ex vivo-activated basophils (CD63(+)/CCR3(+) cells; after stimulation with grass pollen) was substantially lower for idelalisib-treated compared with placebo-treated subjects. Plasma CCL17 and CCL22 levels were reduced after idelalisib treatment. Idelalisib treatment was well tolerated in patients with allergic rhinitis and appears to reduce allergic responses clinically and immunologically after an environmental allergen challenge. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  20. Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms*

    PubMed Central

    Mwaikambo, Bupe R.; Yang, Chun; Chemtob, Sylvain; Hardy, Pierre

    2009-01-01

    Neovascular and degenerative diseases of the eye are leading causes of impaired vision and blindness in the world. Hypoxia or reduced oxygen tension is considered central to the pathogenesis of these disorders. Although the CD36 scavenger receptor features prominently in ocular homeostasis and pathology, little is known regarding its modulation by hypoxia. Herein we investigated the role and regulation of CD36 by hypoxia and by the major hypoxia effector, hypoxia-inducible factor (HIF)-1. In vivo, hypoxia markedly induced CD36 mRNA in corneal and retinal tissue. Subsequent experiments on human retinal pigment epithelial cells revealed that hypoxia time-dependently increased CD36 mRNA, protein, and surface expression; these responses were reliant upon reactive oxygen species production. As an important novel finding, we demonstrate that hypoxic stimulation of CD36 is mediated by HIF-1; HIF-1α down-regulation abolished CD36 induction by both hypoxia and cobalt chloride. Sequence analysis of the human CD36 promoter region revealed a functional HIF-1 binding site. A luciferase reporter construct containing this promoter fragment was activated by hypoxia, whereas mutation at the HIF-1 consensus site decreased promoter activation. Specific binding of HIF-1 to this putative site in hypoxic cells was detected by a chromatin immunoprecipitation assay. Interestingly, inhibition of the phosphatidylinositol 3-kinase pathway blocked the hypoxia-dependent induction of CD36 expression and promoter activity. Functional ramifications of CD36 hypoxic accumulation were evinced by CD36-dependent increases in scavenging and anti-angiogenic activities. Together, our findings indicate a novel mechanism by which hypoxia induces CD36 expression via activation of HIF-1 and the phosphatidylinositol 3-kinase pathway. PMID:19640849

  1. A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis[C][W

    PubMed Central

    Zhuang, Xiaohong; Wang, Hao; Lam, Sheung Kwan; Gao, Caiji; Wang, Xiangfeng; Cai, Yi; Jiang, Liwen

    2013-01-01

    Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain–containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2–green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum–derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis. PMID:24249832

  2. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    PubMed Central

    Leprince, Anne-Sophie; Magalhaes, Nelly; De Vos, Delphine; Bordenave, Marianne; Crilat, Emilie; Clément, Gilles; Meyer, Christian; Munnik, Teun; Savouré, Arnould

    2015-01-01

    Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose. PMID:25628629

  3. Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

    PubMed Central

    Flick, J S; Thorner, J

    1993-01-01

    Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses. Images PMID:8395015

  4. Dynamics of Adipocyte Turnover in Humans

    SciTech Connect

    Spalding, K; Arner, E; Westermark, P; Bernard, S; Buchholz, B; Bergmann, O; Blomqvist, L; Hoffstedt, J; Naslund, E; Britton, T; Concha, H; Hassan, M; Ryden, M; Frisen, J; Arner, P

    2007-07-16

    Obesity is increasing in an epidemic fashion in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. The factors determining fat mass in adult humans are not fully understood, but increased lipid storage in already developed fat cells is thought to be most important. We show that adipocyte number is a major determinant for the fat mass in adults. However, the number of fat cells stays constant in adulthood in lean and obese and even under extreme conditions, indicating that the number of adipocytes is set during childhood and adolescence. To establish the dynamics within the stable population of adipocytes in adults, we have measured adipocyte turnover by analyzing the integration of {sup 14}C derived from nuclear bomb tests in genomic DNA. Approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index. Neither adipocyte death nor generation rate is altered in obesity, suggesting a tight regulation of fat cell number that is independent of metabolic profile in adulthood. The high turnover of adipocytes establishes a new therapeutic target for pharmacological intervention in obesity.

  5. Nitrogen turnover in soil and global change.

    PubMed

    Ollivier, Julien; Töwe, Stefanie; Bannert, Andrea; Hai, Brigitte; Kastl, Eva-Maria; Meyer, Annabel; Su, Ming Xia; Kleineidam, Kristina; Schloter, Michael

    2011-10-01

    Nitrogen management in soils has been considered as key to the sustainable use of terrestrial ecosystems and a protection of major ecosystem services. However, the microorganisms driving processes like nitrification, denitrification, N-fixation and mineralization are highly influenced by changing climatic conditions, intensification of agriculture and the application of new chemicals to a so far unknown extent. In this review, the current knowledge concerning the influence of selected scenarios of global change on the abundance, diversity and activity of microorganisms involved in nitrogen turnover, notably in agricultural and grassland soils, is summarized and linked to the corresponding processes. In this context, data are presented on nitrogen-cycling processes and the corresponding microbial key players during ecosystem development and changes in functional diversity patterns during shifts in land use. Furthermore, the impact of increased temperature, carbon dioxide and changes in precipitation regimes on microbial nitrogen turnover is discussed. Finally, some examples of the effects of pesticides and antibiotics after application to soil for selected processes of nitrogen transformation are also shown.

  6. Relationships between Bone Turnover and Energy Metabolism

    PubMed Central

    2017-01-01

    It is well established that diabetes can be detrimental to bone health, and its chronic complications have been associated with an increased risk of osteoporotic fracture. However, there is growing evidence that the skeleton plays a key role in a whole-organism approach to physiology. The hypothesis that bone may be involved in the regulation of physiological functions, such as insulin sensitivity and energy metabolism, has been suggested. Given the roles of insulin, adipokines, and osteocalcin in these pathways, the need for a more integrative conceptual approach to physiology is emphasized. Recent findings suggest that bone plays an important role in regulating intermediary metabolism, being possibly both a target of diabetic complications and a potential pathophysiologic factor in the disease itself. Understanding the relationships between bone turnover and glucose metabolism is important in order to develop treatments that might reestablish energy metabolism and bone health. This review describes new insights relating bone turnover and energy metabolism that have been reported in the literature. PMID:28695134

  7. Cytoplasmic mRNA turnover and ageing

    PubMed Central

    Borbolis, Fivos; Syntichaki, Popi

    2015-01-01

    Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms. PMID:26432921

  8. Strategies for adapting to high rates of employee turnover.

    PubMed

    Mowday, R T

    1984-01-01

    For many organizations facing high rates of employee turnover, strategies for increasing employee retention may not be practical because employees leave for reasons beyond the control of management or the costs of reducing turnover exceed the benefits to be derived. In this situation managers need to consider strategies that can minimize or buffer the organization from the negative consequences that often follow from turnover. Strategies organizations can use to adapt to uncontrollably high employee turnover rates are presented in this article. In addition, suggestions are made for how managers should make choices among the alternative strategies.

  9. Chicory increases acetate turnover, but not propionate and butyrate peripheral turnovers in rats.

    PubMed

    Pouteau, Etienne; Rochat, Florence; Jann, Alfred; Meirim, Isabelle; Sanchez-Garcia, Jose-Luis; Ornstein, Kurt; German, Bruce; Ballèvre, Olivier

    2008-02-01

    Chicory roots are rich in inulin that is degraded into SCFA in the caecum and colon. Whole-body SCFA metabolism was investigated in rats during food deprivation and postprandial states. After 22 h of food deprivation, sixteen rats received an IV injection of radioactive 14C-labelled SCFA. The volume of distribution and the fractional clearance rate of SCFA were 0.25-0.27 litres/kg and 5.4-5.9 %/min, respectively. The half-life in the first extracellular rapidly decaying compartment was between 0.9 and 1.4 min. After 22 h of food deprivation, another seventeen rats received a primed continuous IV infusion of 13C-labelled SCFA for 2 h. Isotope enrichment (13C) of SCFA was determined in peripheral arterial blood by MS. Peripheral acetate, propionate and butyrate turnover rates were 29, 4 and 0.3 micromol/kg per min respectively. Following 4 weeks of treatment with chicory root or control diets, eighteen fed rats received a primed continuous IV infusion of 13C-labelled SCFA for 2 h. Intestinal degradation of dietary chicory lowered caecal pH, enhanced caecal and colonic weights, caecal SCFA concentrations and breath H2. The diet with chicory supplementation enhanced peripheral acetate turnover by 25 % (P = 0.017) concomitant with an increase in plasma acetate concentration. There were no changes in propionate or butyrate turnovers. In conclusion, by setting up a multi-tracer approach to simultaneously assess the turnovers of acetate, propionate and butyrate it was demonstrated that a chronic chicory-rich diet significantly increases peripheral acetate turnover but not that of propionate or butyrate in rats.

  10. Influence of cortisol, gonadal steroids and an energy deficit on biochemical indicators of bone turnover in Swine.

    PubMed

    Weiler, U; Finsler, S; Claus, R

    2003-03-01

    In the pig a high growth potential seems to favour a disposition for skeletal problems. Hormones of growth hormone (GH)/insulin-like growth factor (IGF)-I axis as well as cortisol and gonadal steroids are endocrine determinants of the anabolic potential but their effects on bone turnover in pigs have not been described. Thus, key hormones were either infused for 7 days (cortisol, 5alpha-dihydrotestosterone (DHT), oestradiol) or influenced by Metyrapone (inhibition of cortisol synthesis) or energy deficit (increasing GH). Each treatment was carried out in six growing barrows/treatment. Bone turnover was characterized by daily measurements indirect parameter of osteoblastic and osteoclastic activity, osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP) respectively. All treatments except cortisol infusion seemed to favour bone formation, as they led either to a pronounced increase in OC (Metyrapone: +14%) or to significantly reduced TRAP (DHT: -9%, E2: -17%, energy deficit: -25%) followed by significantly higher OC (DHT: +9%, E2: +6%, energy deficit: +18%). Cortisol infusion affected bone loss mainly by a severe inhibition of osteoblastic activity (OC: -61%). Some reactions are explained by direct effects of the infused gonadal steroids on bone cells (inhibition of osteoclasts) or of the experimentally modified cortisol levels (inhibition of osteoblasts by cortisol). Other effects seem to be mediated by concomitant changes of IGF-I (inhibition of osteoclasts after energy deficit or cortisol) and GH-secretion (increased osteoblastic activity during energy deficit), respectively. Consequences for co-ordinated bone turnover are discussed.

  11. Turnover intentions and voluntary turnover: the moderating roles of self-monitoring, locus of control, proactive personality, and risk aversion.

    PubMed

    Allen, David G; Weeks, Kelly P; Moffitt, Karen R

    2005-09-01

    This article explores moderators of the relationship between turnover intentions and turnover behavior to better explain why some employees translate intentions into behavior and other employees do not. Individual differences in self-monitoring, locus of control, proactive personality, and risk aversion were examined. Results indicate that self-monitoring and risk aversion moderate the intentions-turnover link. Specifically, the relationship between turnover intentions and turnover is stronger for low self-monitors and those lower in risk aversion. Locus of control moderated the relationship in 1 of 2 samples such that the relationship was stronger for those with an internal locus of control. Proactive personality, however, did not directly moderate the relationship between intentions and turnover behaviors. Copyright 2005 APA, all rights reserved.

  12. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

    SciTech Connect

    Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F. )

    1989-10-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with {sup 3}H-fatty acids, ({sup 3}H)ethanolamine, and ({sup 3}H)carbohydrates. Treatment of {sup 3}H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.

  13. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens.

    PubMed Central

    Tomavo, S; Schwarz, R T; Dubremetz, J F

    1989-01-01

    The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with 3H-fatty acids, [3H]ethanolamine, and [3H]carbohydrates. Treatment of 3H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment. Images PMID:2531282

  14. A complex comprising phosphatidylinositol 4-kinase IIIβ, ACBD3, and Aichi virus proteins enhances phosphatidylinositol 4-phosphate synthesis and is critical for formation of the viral replication complex.

    PubMed

    Ishikawa-Sasaki, Kumiko; Sasaki, Jun; Taniguchi, Koki

    2014-06-01

    Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754-766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornavirus genomes. Aichi virus, a picornavirus belonging to the genus Kobuvirus, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that virus RNA replication

  15. Protein turnover, nitrogen balance and rehabilitation.

    PubMed

    Fern, E B; Waterlow, J C

    1983-01-01

    Not many studies have been done on protein turnover during recovery from malnutrition. Some relevant information can, however, be obtained from measurements on normal growing animals, since rehabilitation and normal growth have in common a rapid rate of net protein synthesis. The key question is the extent to which net gain in protein results from an increase in synthesis or a decrease in breakdown or both. Different studies have used different methods, and all methods for measuring protein turnover have some disadvantages and sources of error. It is important to bear this in mind in evaluating the results. Consequently, part of this paper will be devoted to questions of methodology. Whole body protein turnover has been measured in children recovering from severe malnutrition. During the phase of rapid catch-up growth the rate of protein synthesis is increased. As might be expected, it increases linearly with the rate of weight gain. At the same time there is a smaller increase in the rate of protein breakdown. The resultant of these two processes is that, over and above the basal rate of protein synthesis, 1.4 grams of protein have to be synthesized for 1 gram to be laid down. Very similar results have been obtained in rapidly growing young pigs. Experimental studies on muscle growth in general confirm the conclusion that, at least in muscle, rapid growth is associated with rapid rates of protein breakdown as well as of synthesis. This has been shown in muscles of young growing rats, as well as in muscles in which hypertrophy has been induced by stretch or other stimuli. In contrast, the evidence suggests that rapid growth involves a fall in the rate of protein degradation. The magnitude of the nitrogen balance under any conditions is determined by the difference between synthesis and breakdown. In the absence of any storage of amino acids, this must be the same as the difference between intake and excretion (S - B = I - E). A question of great interest is whether

  16. Turnover of cell surface proteoglycans in cultured fibroblasts

    SciTech Connect

    Brauker, J.H.

    1986-01-01

    Human fibroblasts were cultured in /sup 35/SO/sub 4//sup -2/ to label the glycosaminoglycans (GAGs); the extracellular matrix was derived from these labeled cells by removal of cells with the chelating agent ethylene glycol-bis-(..beta..-amino ethyl ether)N,N'-tetra acetic acid (EGTA). When unlabeled cells were plated onto these radiolabeled extracellular matrices, two distinct events were observed: (a) the cells actively released (/sup 35/S)PG from the matrix and; (b) the cells degraded a large fraction of the matrix PG, releasing free sulfate. The latter degradation event could be inhibited in a specific dose-dependent manner by addition of mannose 6-phosphate to the culture medium. Analyses of this effect in terms of the saccharide specificity, NH/sub 4/Cl sensitivity, and the requirement for cells suggest that both an intracellular compartment and the mannose 6-phosphate receptor that binds lysosomal enzymes at the cell surface may play important roles in the turnover of PG of the extracellular matrix.

  17. Type IIalpha phosphatidylinositol phosphate kinase associates with the plasma membrane via interaction with type I isoforms.

    PubMed Central

    Hinchliffe, Katherine A; Giudici, Maria Luisa; Letcher, Andrew J; Irvine, Robin F

    2002-01-01

    The phosphatidylinositol phosphate kinases (PIPkins) are a family of enzymes involved in regulating levels of several functionally important inositol phospholipids within cells. The PIPkin family is subdivided into three on the basis of substrate specificity, each subtype presumably regulating levels of different subsets of the inositol lipids. The physiological function of the type II isoforms, which exhibit a preference for phosphatidylinositol 5-phosphate, a lipid about which very little is known, is particularly poorly understood. In the present study, we demonstrate interaction between, and co-immunoprecipitation of, type IIalpha PIPkin with the related, but biochemically and immunologically distinct, type I PIPkin isoforms. Type IIalpha PIPkin interacts with all three known type I PIPkins (alpha, beta and gamma), and in each case co-expression of the type I isoform with type IIalpha results in recruitment of the latter from the cytosol to the plasma membrane of the cell. This change in subcellular localization could result in improved access of the type IIalpha PIPkin to its lipid substrates. PMID:11964157

  18. Phosphatidylinositol 4-Phosphate 5-Kinase β Controls Recruitment of Lipid Rafts into the Immunological Synapse.

    PubMed

    Kallikourdis, Marinos; Trovato, Anna Elisa; Roselli, Giuliana; Muscolini, Michela; Porciello, Nicla; Tuosto, Loretta; Viola, Antonella

    2016-02-15

    Phosphatidylinositol 4,5-biphosphate (PIP2) is critical for T lymphocyte activation serving as a substrate for the generation of second messengers and the remodeling of actin cytoskeleton necessary for the clustering of lipid rafts, TCR, and costimulatory receptors toward the T:APC interface. Spatiotemporal analysis of PIP2 synthesis in T lymphocytes suggested that distinct isoforms of the main PIP2-generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), play a differential role on the basis of their distinct localization. In this study, we analyze the contribution of PIP5Kβ to T cell activation and show that CD28 induces the recruitment of PIP5Kβ to the immunological synapse, where it regulates filamin A and lipid raft accumulation, as well as T cell activation, in a nonredundant manner. Finally, we found that Vav1 and the C-terminal 83 aa of PIP5Kβ are pivotal for the PIP5Kβ regulatory functions in response to CD28 stimulation. Copyright © 2016 by The American Association of Immunologists, Inc.

  19. Advances in the researches on the biological activities and inhibitors of phosphatidylinositol 3-kinase.

    PubMed

    Tang, Jian-Feng; Wen, Qing; Sun, Jian; Zhang, Wei-Ming; Zhu, Hai-Liang

    2014-06-01

    The PI3K/AKT/mTOR pathway is an intracellular signaling pathway, being important in apoptosis hence cancer such as breast cancer and non-small-cell lung cancer. It signaling axis controls cell proliferation and survival and has achieved major importance as a target for cancer therapy. The serine/threonine kinase Akt (also known as protein kinase B or PKB), since its initial discovery as a protooncogene, has become a major focus of attention because of its critical regulatory role in diverse cellular processes, including cancer progression and insulin metabolism. The Akt cascade is activated by receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G protein coupled receptors and other stimuli that induce the production of phosphatidylinositol 3,4,5 triphosphates (PtdIns(3,4,5)P3) by phosphoinositide 3-kinase (PI3K). Therefore, PI3K plays an important role in in numerous cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. In this review, we introduced the structure of the PI3K, and then focused on its biological activities. In addition, we reviewed the advances in the researches of PI3K as well as related inhibitors over the last couple of decades. Finally, we also discussed the prospect and developmental trend of phosphatidylinositol 3-kinase as antitumor agents.

  20. Transmembrane Signaling via Phosphatidylinositol 4,5-Bisphosphate Hydrolysis in Plants 1

    PubMed Central

    Einspahr, Kregg J.; Thompson, Guy A.

    1990-01-01

    Recent investigations have confirmed the presence of the polyphosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2), as well as inositol phospholipid-specific phospholipase C in higher plant and microalgal cells. In addition, it has been shown that stimulation of some photosynthetic cell types by environmental or hormonal challenge is accompanied by degradation of the polyphosphoinositides. The products of phospholipase C-catalyzed PIP2 hydrolysis, inositol 1,4,5-trisphosphate and diacylglycerol, appear to be capable of releasing organelle-bound Ca2+ and stimulating protein kinase C-like activity in vitro. However, a direct cause and effect relationship between stimulated PIP2 breakdown and changes in intracellular calcium, protein phosphorylation, or cell function has not yet been unequivocally established. Despite a number of technical difficulties slowing progress in this field, it is likely that photosynthetic organisms will soon be shown to transmit physiologically significant extracellular signals across their plasma membranes by a PIP2-mediated transduction mechanism. PMID:16667474

  1. Localization of Drosophila retinal degeneration B, a membrane- associated phosphatidylinositol transfer protein

    PubMed Central

    1993-01-01

    The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160- kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium. PMID:8354691

  2. Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils

    SciTech Connect

    Eberle, M.; Traynor-Kaplan, A.E.; Sklar, L.A.; Norgauer, J. )

    1990-10-05

    Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated (32P)PIP3 levels decayed toward initial values. However, recovery of (32P)PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of (32P) PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.

  3. Phosphatidylinositol 3-kinase and dynamics of insulin resistance in denervated slow and fast muscles in vivo.

    PubMed

    Elmendorf, J S; Damrau-Abney, A; Smith, T R; David, T S; Turinsky, J

    1997-04-01

    Regulation of glucose uptake by 1- and 3-day denervated soleus (slow-twitch) and plantaris (fast-twitch) muscles in vivo was investigated. One day after denervation, soleus and plantaris muscles exhibited 62 and 65% decreases in insulin-stimulated 2-deoxyglucose uptake, respectively, compared with corresponding control muscles. At this interval, denervated muscles showed no alterations in insulin receptor binding and activity, amount and activity of phosphatidylinositol 3-kinase, and amounts of GLUT-1 and GLUT-4. Three days after denervation, there was no increase in 2-deoxyglucose uptake in response to insulin in soleus muscle, whereas plantaris muscle exhibited a 158% increase in basal and an almost normal absolute increment in insulin-stimulated uptake. Despite these differences, denervated soleus and plantaris muscles exhibited comparable decreases in insulin-stimulated activities of the insulin receptor (approximately 40%) and phosphatidylinositol 3-kinase (approximately 50%) and a pronounced decrease in GLUT-4. An increase in GLUT-1 in plantaris, but not soleus, muscle 3 days after denervation is consistent with augmented basal 2-deoxyglucose uptake in plantaris muscle at this interval. These results demonstrate that, in denervated muscles, there is a clear dissociation between insulin-stimulated 2-deoxyglucose uptake and upstream events involved in insulin-stimulated glucose uptake.

  4. Analysis of glycosyl phosphatidylinositol-anchored proteins by two-dimensional gel electrophoresis.

    PubMed

    Fivaz, M; Vilbois, F; Pasquali, C; van der Goot, F G

    2000-10-01

    The aim of this study was to characterize mammalian glycosyl phosphatidylinositol (GPI)-anchored proteins y two-dimensional gel electrophoresis using immobilized pH gradients. Analysis was performed on detergent-resistant membrane fractions of baby hamster kidney (BHK) cells, since such fractions have previously been shown to be highly enriched in GPI-anchored proteins. Although the GPI-anchored proteins were readily separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these proteins were undetectable on two-dimensional (2-D) gels, even though these gels unambiguously revealed high enrichment of known hydrophobic proteins of detergent-resistant membranes such as caveolin-1 and flotillin-1 (identified by Western blotting and tandem mass spectrometry, respectively). Proper separation of GPI-anchored proteins required cleavage of the lipid tail with phosphatidylinositol-specific phospholipase C, presumably to avoid interference of the hydrophobic phospholipid moiety of GPI-anchors during isoelectric focusing. Using this strategy, BHK cells were observed to contain at least six GPI-anchored proteins. Each protein was also present as multiple isoforms with different isoelectric points and apparent molecular weights, consistent with extensive but differential N-glycosylation. Pretreatment with N-glycosidase F indeed caused the different isoforms of each protein to collapse into a single spot. In addition, quantitative removal of N-linked sugars greatly facilitated the detection of heavily glycosylated proteins and enabled sequencing by nanoelectrospray-tandem mass spectrometry as illustrated for the GPI-anchored protein, Thy-1.

  5. Two phosphatidylinositol 4-kinases control lysosomal delivery of the Gaucher disease enzyme, β-glucocerebrosidase

    PubMed Central

    Jović, Marko; Kean, Michelle J.; Szentpetery, Zsofia; Polevoy, Gordon; Gingras, Anne-Claude; Brill, Julie A.; Balla, Tamas

    2012-01-01

    Gaucher disease is a lysosomal storage disorder caused by a defect in the degradation of glucosylceramide catalyzed by the lysosomal enzyme β-glucocerebrosidase (GBA). GBA reaches lysosomes via association with its receptor, lysosomal integral membrane protein type 2 (LIMP-2). We found that distinct phosphatidylinositol 4-kinases (PI4Ks) play important roles at multiple steps in the trafficking pathway of the LIMP-2/GBA complex. Acute depletion of phosphatidylinositol 4-phosphate in the Golgi caused accumulation of LIMP-2 in this compartment, and PI4KIIIβ was found to be responsible for controlling the exit of LIMP-2 from the Golgi. In contrast, depletion of PI4KIIα blocked trafficking at a post-Golgi compartment, leading to accumulation of LIMP-2 in enlarged endosomal vesicles. PI4KIIα depletion also caused secretion of missorted GBA into the medium, which was attenuated by limiting LIMP-2/GBA exit from the Golgi by PI4KIIIβ inhibitors. These studies identified PI4KIIIβ and PI4KIIα as important regulators of lysosomal delivery of GBA, revealing a new element of control to sphingolipid homeostasis by phosphoinositides. PMID:22337770

  6. Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases?

    PubMed

    Mikami, Koji; Saavedra, Laura; Sommarin, Marianne

    2010-10-01

    Phosphatidylinositol phosphate kinase (PIPK) catalyzes a key step controlling cellular contents of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], a critical intracellular messenger involved in vesicle trafficking and modulation of actin cytoskeleton and also a substrate of phospholipase C to produce the two intracellular messengers, diacylglycerol and inositol-1,4,5-trisphosphate. In addition to the conserved C-terminal PIPK catalytic domain, plant PIPKs contain a unique structural feature consisting of a repeat of membrane occupation and recognition nexus (MORN) motifs, called the MORN domain, in the N-terminal half. The MORN domain has previously been proposed to regulate plasma membrane localization and phosphatidic acid (PA)-inducible activation. Recently, the importance of the catalytic domain, but not the MORN domain, in these aspects was demonstrated. These conflicting data raise the question about the function of the MORN domain in plant PIPKs. We therefore performed analyses of PpPIPK1 from the moss Physcomitrella patens to elucidate the importance of the MORN domain in the control of enzymatic activity; however, we found no effect on either enzymatic activity or activation by PA. Taken together with our previous findings of lack of function in plasma membrane localization, there is no positive evidence indicating roles of the MORN domain in enzymatic and functional regulations of PpPIPK1. Therefore, further biochemical and reverse genetic analyses are necessary to understand the biological significance of the MORN domain in plant PIPKs.

  7. Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses*♦

    PubMed Central

    Rhee, Hong Jun; Subramanian, Devaraj; Paraskevopoulou, Foteini; Mueller, Rainer; Schultz, Carsten; Brose, Nils; Rhee, Jeong-Seop; Betz, Heinrich

    2017-01-01

    The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, time-lapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/sorting endosomes regulates the postsynaptic clustering of gephyrin and GABAA receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cb-mediated recruitment of gephyrin and GABAA receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations. PMID:27941024

  8. Chemical Synthesis and Molecular Recognition of Phosphatase-Resistant Analogues of Phosphatidylinositol-3-phosphate

    PubMed Central

    Xu, Yong; Lee, Stephanie A.; Kutateladze, Tatiana G.; Sbrissa, Diego; Shisheva, Assia; Prestwich, Glenn D.

    2008-01-01

    The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. In order to provide chemical tools to study of the changes in cell physiology mediated by these lipids, three new metabolically-stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-monofluoromethylphosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl and dibutyryl chains. In addition, we introduce a new phosphorlyation reagent, monofluoromethylphosphonyl chloride, which has general applications for the preparation of “pKa-matched” monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labelled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)Ps into membrane-mimetic dodecylphosphocholine (DPC) micelles. In addition, the PtdIns(3)P analogues with dioleyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis. PMID:16417379

  9. Protein Turnover during in vitro Tissue Engineering

    PubMed Central

    Li, Qiyao; Chang, Zhen; Oliveira, Gisele; Xiong, Maiyer; Smith, Lloyd M.; Frey, Brian L.; Welham, Nathan V.

    2015-01-01

    Repopulating acellular biological scaffolds with phenotypically appropriate cells is a promising approach for regenerating functional tissues and organs. Under this tissue engineering paradigm, reseeded cells are expected to remodel the scaffold by active protein synthesis and degradation; however, the rate and extent of this remodeling remain largely unknown. Here, we present a technique to measure dynamic proteome changes during in vitro remodeling of decellularized tissue by reseeded cells, using vocal fold mucosa as the model system. Decellularization and recellularization were optimized, and a stable isotope labeling strategy was developed to differentiate remnant proteins constituting the original scaffold from proteins newly synthesized by reseeded cells. Turnover of matrix and cellular proteins and the effects of cell-scaffold interaction were elucidated. This technique sheds new light on in vitro tissue remodeling and the process of tissue regeneration, and is readily applicable to other tissue and organ systems. PMID:26724458

  10. Teacher Turnover, Teacher Quality, and Student Achievement in DCPS

    ERIC Educational Resources Information Center

    Adnot, Melinda; Dee, Thomas; Katz, Veronica; Wyckoff, James

    2017-01-01

    In practice, teacher turnover appears to have negative effects on school quality as measured by student performance. However, some simulations suggest that turnover can instead have large positive effects under a policy regime in which low-performing teachers can be accurately identified and replaced with more effective teachers. This study…

  11. How Multiple Interventions Influenced Employee Turnover: A Case Study.

    ERIC Educational Resources Information Center

    Hatcher, Timothy

    1999-01-01

    A 3-year study of 46 textile industry workers identified causes of employee turnover (supervision, training, organizational communication) using performance analysis. A study of multiple interventions based on the analysis resulted in changes in orientation procedures, organizational leadership, and climate, reducing turnover by 24%. (SK)

  12. Creating a nursing residency: decrease turnover and increase clinical competence.

    PubMed

    Welding, Nicole M

    2011-01-01

    New graduates are the largest source of registered nurses available for recruitment, and graduates are expected to transition quickly into professional practice. Stress of this transition can lead to high turnover within the first year. The design and goals of a graduate nurse residency program to increase competence, leadership, and job satisfaction, and ultimately decrease turnover are reported.

  13. The Link between Training Satisfaction, Work Engagement and Turnover Intention

    ERIC Educational Resources Information Center

    Memon, Mumtaz Ali; Salleh, Rohani; Baharom, Mohamed Noor Rosli

    2016-01-01

    Purpose: The purpose of this paper is to examine the casual relationship between training satisfaction, work engagement (WE) and turnover intention and the mediating role of WE between training satisfaction and turnover intention. Design/methodology/approach: Data were collected from 409 oil and gas professionals using an email survey…

  14. Re-Examining the Relationship between Age and Voluntary Turnover

    ERIC Educational Resources Information Center

    Ng, Thomas W. H.; Feldman, Daniel C.

    2009-01-01

    In their quantitative review of the literature, Healy, Lehman, and McDaniel [Healy, M. C., Lehman, M., & McDaniel, M. A. (1995). Age and voluntary turnover: A quantitative review. "Personnel Psychology, 48", 335-345] concluded that age is only weakly related to voluntary turnover (average r = -0.08). However, with the significant changes in…

  15. Predicting Turnover: Validating the Intent to Leave Child Welfare Scale

    ERIC Educational Resources Information Center

    Auerbach, Charles; Schudrich, Wendy Zeitlin; Lawrence, Catherine K.; Claiborne, Nancy; McGowan, Brenda G.

    2014-01-01

    A number of proxies have been used in child welfare workforce research to represent actual turnover; however, there have been no psychometric studies to validate a scale specifically designed for this purpose. The Intent to Leave Child Welfare Scale is a proxy for actual turnover that measures workers' intention to leave. This scale was validated…

  16. Analysis of the Educational Personnel System: IV. Teacher Turnover.

    ERIC Educational Resources Information Center

    Keeler, Emmett B.

    This report attempts to predict the rates of teacher turnover in the 1970s, which teachers will leave the profession, and what the effects of turnover will be on the educational personnel system. The overall termination rate has varied from six to 11 percent over the last 15 years. An analysis of recent changes in the teaching profession is used…

  17. How Multiple Interventions Influenced Employee Turnover: A Case Study.

    ERIC Educational Resources Information Center

    Hatcher, Timothy

    1999-01-01

    A 3-year study of 46 textile industry workers identified causes of employee turnover (supervision, training, organizational communication) using performance analysis. A study of multiple interventions based on the analysis resulted in changes in orientation procedures, organizational leadership, and climate, reducing turnover by 24%. (SK)

  18. Organizational Characteristics Associated with Staff Turnover in Nursing Homes

    ERIC Educational Resources Information Center

    Castle, Nicholas G.; Engberg, John

    2006-01-01

    Purpose: The association between certified nurse aide, licensed practical nurse, and registered nurse turnover and the organizational characteristics of nursing homes are examined. Design and Methods: Hypotheses for eight organizational characteristics are examined (staffing levels, top management turnover, resident case mix, facility quality,…

  19. Re-Examining the Relationship between Age and Voluntary Turnover

    ERIC Educational Resources Information Center

    Ng, Thomas W. H.; Feldman, Daniel C.

    2009-01-01

    In their quantitative review of the literature, Healy, Lehman, and McDaniel [Healy, M. C., Lehman, M., & McDaniel, M. A. (1995). Age and voluntary turnover: A quantitative review. "Personnel Psychology, 48", 335-345] concluded that age is only weakly related to voluntary turnover (average r = -0.08). However, with the significant changes in…