Level Of Pi Research Articles

Abstract WP209: Serum Anti-Periodontal Pathogen Antibody associated with Atrial Fibrillation and Carotid Plaque

Background: We evaluated whether serum antibody levels against individual periodontal pathogens are significantly associated with ischemic stroke subtypes and their risk factors. Methods: Patients with acute ischemic stroke (n=132; 74 male and 58 female, 71.3±10.7 years) and patients with no previous stroke (n=77; 38 male and 39 female, 70.7±9.5 years) were consecutively enrolled in this study. Stroke subtype was evaluated based on the TOAST classification. Serum was obtained from each patient after obtaining the patient’s consent to participate in this study. The levels of serum antibodies against Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Prevotella intermedia (Pi) were evaluated by ELISA. Results: The serum-antibody level of Pi was significantly higher in atherothrombotic-stroke patients than in patients with no previous stroke (p<0.005). Serum anti-Pg antibody was significantly associated with atrial fibrillation (overall X2=35.5, R2=0.18, n=209, p<0.001; anti-Pg antibody: odds ratio 4.36 [95%CI 1.71-12.10], p<0.005), and serum anti-Pi antibody was significantly associated with bulb/internal carotid artery atherosclerosis after controlling for the statistically selected associated factors (overall X2=46.1, R2=0.18, n=209, p<0.001; anti-Pg antibody: odds ratio 16.58 [95%CI 3.96-78.93], p<0.001). The levels of serum anti-Pi antibody were significantly associated with atherothrombotic stroke with the statistically selected associated factors excluding bulb/ICA atherosclerosis (overall X2=77.0, R2=0.44, n=129, p<0.001; anti-Pi antibody: odds ratio 23.6 [95%CI 2.65-298.2], p=0.008). However, when we included bulb/ICA atherosclerosis into this model, the levels of serum anti-Pi antibody were no longer significantly associated with atherothrombotic stroke (overall X2=98.0, R2=0.56, n=129, p<0.0001; anti-Pi antibody: p=0.107). Conclusions: Our results suggest that anti-Pg antibody is associated with atrial fibrillation and anti-Pi antibody is associated with carotid artery atherosclerosis. Additionally, anti-Pi antibody may be associated with atherothrombotic stroke through its association with carotid artery atherosclerosis.

The Effect of Phosphate on the Force Generating Capacity of a Mini-Ensemble of Myosin Measured in a Laser Trap Assay

The accumulation of inorganic phosphate (Pi) is believed to cause much of the loss in the force-generating capacity of muscle during fatigue, but the molecular basis of this effect remains poorly understood. To gain insight into this process we determined the effect of a fatiguing level of Pi (30mM) on the force-generating capacity of a mini-ensemble of skeletal myosin (∼8molecules) interacting with a single actin filament using a three bead laser trap assay, at 100uM ATP. In the absence of Pi the average force generated displayed a bimodal Gaussian distribution with a low force peak centered at 0.30 ± 0.02 pN and second higher peak centered at 1.22 ± 0.08 pN. In the presence of Pi a low force peak centered at 0.28 ± 0.02 pN remained but the high force peak was completely absent. The duration of the low force binding events were statistically similar in both the presence (30 ± 4ms) and absence of Pi (28 ± 3ms) as were the average displacements of these events at 14 ± 1nm and 16 ± 1nm respectively, consistent with a mix of single and double molecule binding events. The high force events generated on average 64 ± 4nm displacements lasting 75 ± 8ms, suggesting these resulted from multiple binding events before detachment from the actin filament. The overall frequency of binding events was similar in the presence (3.0/s) and absence of Pi (2.7s). Taken together these data suggest that Pi reduces the force-generating capacity of a myosin by eliminating the ability to make the successive and simultaneous actin attachments required to generate higher forces.

Association of Serum Anti-Periodontal Pathogen Antibody with Ischemic Stroke

Background: Periodontitis increases the risk of atherosclerotic cardiovascular disease and ischemic stroke. In this study, we evaluated whether serum antibody levels against individual periodontal pathogens are significantly associated with ischemic stroke subtypes and their risk factors. Methods: Patients with acute ischemic stroke (n = 132; 74 male and 58 female, 71.3 ± 10.7 years) and patients with no previous stroke (n = 77; 38 male and 39 female, 70.7 ± 9.5 years) were consecutively enrolled in this study. Stroke subtype was evaluated based on the Trial of Org 10172 in Acute Stroke Treatment classification. Serum was obtained from each patient after obtaining their consent to participate in the study. The levels of serum antibodies against Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi) were evaluated by ELISA. Serum high-sensitivity C-reactive protein (hs-CRP) levels were measured by nephelometry. Results: Serum hs-CRP levels were significantly associated with acute ischemic stroke even after controlling for acute ischemic stroke, hypertension, diabetes mellitus and bulb/ internal carotid artery (ICA) atherosclerosis which were statistically selected (coefficient 0.245, 95% CI 0.142–0.347, p < 0.0001). The serum-antibody level of Pi was significantly higher in atherothrombotic-stroke patients than in patients with no previous stroke (p = 0.0035). Detectable serum anti-Pg antibody was significantly associated with atrial fibrillation (overall χ² = 35.5, R² = 0.18, n = 209, p < 0.0001; anti-Pg antibody: OR 4.36, 95% CI 1.71–12.10, p = 0.0017), and detectable serum anti-Pi antibody was significantly associated with bulb/ICA atherosclerosis after controlling for the statistically selected associated factors (overall χ² = 46.1, R² = 0.18, n = 209, p < 0.0001; anti-Pg antibody: OR 16.58, 95% CI 3.96–78.93, p < 0.0001). The levels of serum anti-Pi antibody were significantly associated with atherothrombotic stroke with the statistically selected associated factors excluding bulb/ICA atherosclerosis (overall χ² = 77.0, R² = 0.44, n = 129, p < 0.0001; anti-Pi antibody: OR 23.6, 95% CI 2.65–298.2, p = 0.008). However, when we included bulb/ICA atherosclerosis in this model, the levels of serum anti-Pi antibody were no longer significantly associated with atherothrombotic stroke (overall χ² = 98.0, R² = 0.56, n = 129, p < 0.0001; anti-Pi antibody: p = 0.107). Conclusions: Our results suggest that anti-Pg antibody is associated with atrial fibrillation and that anti-Pi antibody is associated with carotid artery atherosclerosis. In addition, anti-Pi antibody may be associated with atherothrombotic stroke through its association with carotid artery atherosclerosis. Thus, periodontitis may lead to serious systemic diseases.

SHIP2 signalling at the plasma membrane, in the nucleus and at focal contacts

Phosphoinositide 5-phosphatases are critical enzymes in modulating the concentrations of PI(3,4,5)P(3), PI(4,5)P(2) and PI(3,5)P(2). The SH2 domain containing inositol 5-phosphatases SHIP1 and SHIP2 belong to this family of enzymes very much involved in physiopathology and development. Therefore activity and localization of the enzymes are particularly important taking into account both catalytic and non-catalytic mechanisms of the SHIP phosphatases. Several different mechanisms have been reported for SHIP2 targeting that often result from specific protein:protein interactions. In unstimulated astrocytoma cells, SHIP2 has a perinuclear and cytoplasmic localization. In serum-stimulated cells, SHIP2 can be localized at the plasma membrane and at focal contacts in polarized cells. A phosphorylated form of SHIP2 on S132 can be found in the nucleus and nuclear speckles. When present at the plasma membrane, SHIP2 may control the intracellular level of PI(3,4,5)P(3) thereby producing PI(3,4)P(2). When present in the nucleus, SHIP2 probably associates to other nuclear proteins such as lamin A/C and could potentially control nuclear PI(4,5)P(2). Finally, its presence at focal adhesions and lamellipodia could suggest a role in cell adhesion and migration. It is proposed that the complex phenotype observed in SHIP2 mutant mice in tissue development and growth could result from the addition of plasma membrane and nuclear effects consecutive to SHIP2 alteration.

Effects of geranyl-phloroacetophenone on the induction of apoptosis and chemosensitization of adriamycin-resistant MCF-7 human breast cancer cells

Polyphenols are known to induce apoptosis in many cancer cells and are proposed to be promising modulators of drug resistance. In the present study, we report that 3-geranyl-phloroacetophenone (3-GAP), a synthetic polyphenol, induces apoptosis and modulates drug resistance. In adriamycin-resistant MCF-7 human breast cancer (MCF-7/ADR) cells, which express a mutant form of p53, 3-GAP induced significant apoptosis, which was accompanied by no change in p53 transcriptional activity, but an increase in Bax expression, cyt c release, and activation of caspase-9, 7, and 3. In addition, 3-GAP significantly decreased the activity and expression level of glutathione S-transferase pi (GSTπ), a factor that induces drug resistance. Along with GSTπ inhibition, 3-GAP also induced a marked depletion of GSH, an endogenous antioxidant. The GST-inhibitory activity of 3-GAP correlated with the sensitization of MCF-7/ADR cells to doxorubicin. Under serum withdrawal conditions, the JNK inhibitor SP600125 significantly decreased the viability of the parent MCF-7 cells but not of MCF-7/ADR cells. In addition, the viability of 3-GAP-treated MCF-7/ADR cells was similar to those of MCF-7 cells treated with SP600125 alone or MCF-7/ADR cells co-treated with SP600125 and 3-GAP. These results indicate that JNK activity in MCF-7/ADR cells is halted by high levels of GSTπ, and that 3-GAP releases JNK from GSTπ's inhibition. In conclusion, 3-GAP induces apoptosis in and sensitizes drug-resistant MCF-7/ADR cells. These effects are mediated through p53-independent caspase-3 activation and reduction of the capacity for cellular antioxidants, such as GSTπ and GSH.

Zoledronate inhibits phosphate and bone morphogenetic protein 2-induced extracellular calcification of vascular smooth muscle cells in vitro

The aim of this study was to explore the effects of the bisphosphonate zoledronate on calcification induced by inorganic phosphate (Pi) and/or bone morphogenetic protein 2 (BMP-2) and the underlying mechanisms. Primary vascular smooth muscle cells (VSMCs) from rats were treated with 3 mM Pi or 3 mM Pi/BMP-2, with and without addition of zoledronate; 1.4 mM Pi served as a control. Calcium deposits, expression of core binding factor α-1 (Cbfa-1), osteopontin (OPN), parathyroid pituitary-specific transcription factor (Pit)-1 and Pit-2, and Pi uptake of VSMCs was determined. The calcification of VSMCs induced by elevated Pi or Pi/BMP-2 was significantly inhibited by zoledronate. The expression of Cbfa-1, OPN and Pit-1 was increased significantly after treatment with an elevated level of Pi or Pi/BMP-2, and this expression was significantly suppressed by addition of zoledronate. Pi uptake of VSMCs increased following treatment with elevated Pi and significantly decreased by addition of zoledronate. These results indicated that zoledronate effectively inhibited calcification induced by Pi/BMP-2, and this may have been achieved by means of the downregulation of expression of calcification-related proteins and uptake of Pi.

Simulating virtual environments within virtual environments as the basis for a psychophysics of presence

A new definition of immersion with respect to virtual environment (VE) systems has been proposed in earlier work, based on the concept of simulation. One system (A) is said to be more immersive than another (B) if A can be used to simulate an application as if it were running on B. Here we show how this concept can be used as the basis for a psychophysics of presence in VEs, the sensation of being in the place depicted by the virtual environment displays (Place Illusion, PI), and also the illusion that events occurring in the virtual environment are real (Plausibility Illusion, Psi). The new methodology involves matching experiments akin to those in color science. Twenty participants first experienced PI or Psi in the initial highest level immersive system, and then in 5 different trials chose transitions from lower to higher order systems and declared a match whenever they felt the same level of PI or Psi as they had in the initial system. In each transition they could change the type of illumination model used, or the field-of-view, or the display type (powerwall or HMD) or the extent of self-representation by an avatar. The results showed that the 10 participants instructed to choose transitions to attain a level of PI corresponding to that in the initial system tended to first choose a wide field-of-view and head-mounted display, and then ensure that they had a virtual body that moved as they did. The other 10 in the Psi group concentrated far more on achieving a higher level of illumination realism, although having a virtual body representation was important for both groups. This methodology is offered as a way forward in the evaluation of the responses of people to immersive virtual environments, a unified theory and methodology for psychophysical measurement.

Simulating virtual environments within virtual environments as the basis for a psychophysics of presence

A new definition of immersion with respect to virtual environment (VE) systems has been proposed in earlier work, based on the concept of simulation. One system ( A ) is said to be more immersive than another ( B ) if A can be used to simulate an application as if it were running on B. Here we show how this concept can be used as the basis for a psychophysics of presence in VEs, the sensation of being in the place depicted by the virtual environment displays (Place Illusion, PI), and also the illusion that events occurring in the virtual environment are real (Plausibility Illusion, Psi). The new methodology involves matching experiments akin to those in color science. Twenty participants first experienced PI or Psi in the initial highest level immersive system, and then in 5 different trials chose transitions from lower to higher order systems and declared a match whenever they felt the same level of PI or Psi as they had in the initial system. In each transition they could change the type of illumination model used, or the field-of-view, or the display type (powerwall or HMD) or the extent of self-representation by an avatar. The results showed that the 10 participants instructed to choose transitions to attain a level of PI corresponding to that in the initial system tended to first choose a wide field-of-view and head-mounted display, and then ensure that they had a virtual body that moved as they did. The other 10 in the Psi group concentrated far more on achieving a higher level of illumination realism, although having a virtual body representation was important for both groups. This methodology is offered as a way forward in the evaluation of the responses of people to immersive virtual environments, a unified theory and methodology for psychophysical measurement.

Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells

Human immunodeficiency virus type 1 (HIV-1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV-1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV-1-infected primary CD4(+) T-cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 2/3 of Tat are exported by HIV-1-infected cells during their lifespan. The function of extracellular Tat in HIV-1 infection might thus be more significant than earlier thought.

Local Regulation of Free PI(4,5)P2 Levels by MARCKS Protein

The lipid phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) has an important role in regulating membrane‐cytoskeleton‐adhesion and many other cellular processes. Although synthesis and degradation may locally regulate levels of PI(4,5)P2 in many cases, an alternative mechanism could be buffering by cationic proteins, like MARCKS . We found that increasing MARCKS protein levels by about 50% caused a decrease in membrane‐cytoskeleton adhesion by two‐fold, consistent with a drop in free PI(4,5) P2 levels. The drop in PI(4,5) P2 levels was reversed by over‐expression of phosphatidylinositol phosphate 5‐kinase type Iα (PIP5KIα) or by phosphorylation of MARCKS by PKC activation. Biochemical analyses of MARCKS‐/‐ fibroblasts showed elevated rates of turnover of both PI(4)P and PI(4,5)P2 as well as a decrease in PA turnover. Although total cellular level of PI(4,5)P2 did not change in MARCKS‐/‐ cells, the plasma membrane level of PI(4,5)P2 decreased by two fold, consistent with the loss of a buffer. All of these data suggest that there is compartmental regulation of PI(4,5)P2 in the cell that is not only controlled by the balance between synthesis and degradation, but also via sequestration by MARCKS protein.

High external phosphate (Pi) increases sodium ion uptake and reduces salt tolerance of ‘Pi‐tolerant’ soybean

Previous studies on the interaction between environmental inorganic phosphate (Pi) and salinity stress using soybean cultivars sensitive to high external Pi had two limitations: (1) the phenotype was dominated by overaccumulation of phosphorus (P); and (2) no detailed analysis was performed for sodium ion uptake. In this study, we focused on the effects of high external Pi on the sodium ion uptake in 'Pi-tolerant' soybean cultivars. The P accumulation in Pi-tolerant soybean Union was much lower [9.0 mg g(-)(1) dry weight (DW); contrasting to 38-76 mg g(-)(1) DW in the 'Pi-sensitive' soybean cultivars]. At in planta level, high level of external Pi significantly (P < 0.001) increased net sodium ion uptake and aggravated salinity stress symptoms. The effects of high external Pi diminished when de-rooted plants were used, suggesting that root is the primary organ interacting with Pi in the growth medium. Two-cell models, including soybean suspension cells and the tobacco Bright Yellow-2 cell line, were also employed to study the effects of high external Pi at the cellular level. Consistent to in planta results, high external Pi uplifted cellular sodium ion uptake and reduced cell viability under salinity stress. Gene expression analyses further showed that HPi (2 mM Pi supplements; excessive level of Pi) could reduce the fold of induction of GmSOS1 and GmCNGC under salinity stress, suggesting that they may be possible molecular targets involved in the interaction between high external Pi and Na(+) uptake.

Kinetic metabolic modelling for the control of plant cells cytoplasmic phosphate

A previously developed kinetic metabolic model for plant metabolism was used in a context of identification and control of intracellular phosphate (Pi) dynamics. Experimental data from batch flask cultures of Eschscholtiza californica cells was used to calibrate the model parameters for the slow dynamics (growth, nutrition, anabolic pathways, etc.). Perturbation experiments were performed using a perfusion small-scale bioreactor monitored by in vivo 31P NMR. Parameter identification for Pi metabolism was done by measuring the cells dynamic response to different inputs for extracellular Pi (two pulse-response experiments and a step-response experiment). The calibrated model can describe Pi translocation between the cellular pools (vacuole and cytoplasm). The effect of intracellular Pi management on ATP/ADP and phosphomonoesters concentrations is also described by the model. The calibrated model is then used to develop a control strategy on the cytoplasmic Pi pool. From the identification of the systems dynamics, a proportional-integral controller was designed and tuned. The closed-loop control was implemented in the small-scale NMR bioreactor and experimental results were in accordance with model predictions. Thus, the calibrated model is able to predict cellular behaviour for phosphate metabolism and it was demonstrated that it is possible to control the intracellular level of cytoplasmic Pi in plant cells.

Hepatic and extrahepatic expression of glutathione S-transferase isozymes in mice and its modulation by naturally occurring phenolic acids

A simple plant phenolic acid, protocatechuic acid and a polyphenol, tannic acid are potential chemopreventive agents which inhibited the chemically induced carcinogenesis in many experimental models. We previously demonstrated that those compounds modulate the activity of xenobiotic detoxifying enzymes, including GST in mouse liver, kidney and epidermis. Intraperitoneal (i.p.) treatment with protocatechuic acid in the dose of 80 mg/kg for three consecutive days increased the GST activity in liver and kidney. In case of tannic acid the same effect was observed in kidney after i.p. administration of the single dose of 80 mg/kg. Topical application of phenolic acids resulted in enhancement of epidermal GST activity. The focus of this study was to further investigate the effects of these phenolic acids on the protein levels of GST isozymes in the same tissues using the treatment protocols used in our previous studies. The results confirmed the expression of GST alfa, mu, pi and theta in mouse liver, kidney and epidermis. Treatment with protocatechuic acid resulted in an increase of the expression of GST class mu in liver, but did not affect this isoform in skin and kidney. This compound inhibited the level of kidney GST pi by 35%. Tannic acid decreased the expression of GST mu, alpha and theta in liver. Application of the equimolar doses of both phenolic acids on mouse skin resulted in reduced level of the GST alpha protein. The results of our study indicate that, although moderate, the effect of protocatechuic acid and tannic acid on GST subunits in mice may play certain role in biological activity of these compounds. Of special importance could be the increased expression of GST mu in liver which is involved in detoxification of many carcinogens including polycyclic aromatic hydrocarbons.

Type Iγ661 Phosphatidylinositol Phosphate Kinase Directly Interacts with AP2 and Regulates Endocytosis

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.

Evaluation of MR signal index for the assessment of occupational manganese exposure of welders by measurement of local proton T1 relaxation time

Magnetic resonance (MR) imaging has become a useful tool for the estimation of occupational exposure to manganese (Mn). However, validity of signal indices on T 1-weighted imaging, a proxy measurement of T 1 relaxation time shortening due to Mn deposition, needs to be evaluated in an occupationally exposed population. We performed MR imaging on 20 male welders and 10 age- and gender-matched, non-office workers in a shipyard. We measured the relative signal intensity ratio of the globus pallidus (GP) to the frontal white matter on T 1-weighted MR images (pallidal index, PI). We also assessed the visual scale for the relative signal intensity of the GP, which was graded as 0–2 by a radiologist. We evaluated both signal index and visual scale in relation to the T 1 relaxation time measured at the GP. Both PI and T 1 relaxation time were significantly increased in the welders compared to the non-welders. T 1 relaxation time began to decrease from visual scale 2, while PI began to increase from visual scale 1. Environmental Mn exposure indices correlated well with both T 1 relaxation time and PI. However, blood Mn level correlated only with T 1 relaxation time, and not with PI. T 1 relaxation time correlated with PI only at the higher level of PI. These results indicate the possible inaccuracy of visual scoring and the discrepancy between T 1 relaxation time and PI, especially at the lower level of signal intensity. Although both T 1 and PI are good biomarkers for the environmental Mn exposure, non-linearity of the relationship between T 1 and PI must be considered in the assessment of Mn exposure using MRI.

The composition of pulmonary surfactant from diving mammals

Maintaining a functional pulmonary surfactant system at depth is critical for diving mammals to ensure that inspiration is possible upon re-emergence. The lipid and protein composition of lavage extracts from three pinniped species (California sea lion, Northern elephant seal and Ringed seal) were compared to several terrestrial species. Lavage samples were purified using a NaBr discontinuous gradient. Concentrations of phospholipid classes and molecular species were measured using electrospray ionisation mass spectrometry, cholesterol was measured using high-performance liquid chromatography, surfactant protein A (SP-A) and SP-B were measured using enzyme-linked immunosorbent assays. There were small differences in phospholipid classes, with a lower level of anionic surfactant phospholipids, PG and PI, between diving and terrestrial mammals. There were no differences in PL saturation or SP-A levels between species. PC16:0/14:0, PC16:0/16:1, PC16:0/16:0, long chain PI species and the total concentrations of alkyl-acyl species of PC and PG as a ratio of diacyl species were increased in diving mammals, whereas concentrations of PC16:0/18:1, PG16:0/16:0 and PG16:0/18:1 were decreased. Cholesterol levels were very variable between species and SP-B was very low in diving mammals. These differences may explain the very poor surface activity of pinniped surfactant that we have previously described [Miller, N.J., Daniels, C.B., Schürch, S., Schoel, W.M., Orgeig, S., 2005. The surface activity of pulmonary surfactant from diving mammals. Respir. Physiol. Neurobiol. 150 (2006) 220-232], supporting the hypothesis that pinniped surfactant has primarily an anti-adhesive function to meet the challenges of regularly collapsing lungs.

The LIM Protein Ajuba Regulates Phosphatidylinositol 4,5-Bisphosphate Levels in Migrating Cells through an Interaction with and Activation of PIPKIα

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many actin-binding proteins and as such is an important modulator of cytoskeleton organization during cell migration, for example. In migrating cells actin remodeling is tightly regulated and localized; therefore, how the PI(4,5)P2 level is spatially and temporally regulated is crucial to understanding how it controls cell migration. Here we show that the LIM protein Ajuba contributes to the cellular regulation of PI(4,5)P2 levels by interacting with and activating the enzymatic activity of the PI(4)P 5-kinase (PIPKIalpha), the predominant enzyme in the synthesis of PI(4,5)P2, in a migration stimulus-regulated manner. In migrating primary mouse embryonic fibroblasts (MEFs) from Ajuba(-/-) mice the level of PI(4,5)P2 was decreased with a corresponding increase in the level of the substrate PI(4)P. Reintroduction of Ajuba into these cells normalized PI(4,5)P2 levels. Localization of PI(4,5)P2 synthesis and PIPKIalpha in the leading lamellipodia and membrane ruffles, respectively, of migrating Ajuba(-/-) MEFs was impaired. In vitro, Ajuba dramatically activated the enzymatic activity of PIPKIalpha while inhibiting the activity of PIPKIIbeta. Thus, in addition to its effects upon Rac activity Ajuba can also influence cell migration through regulation of PI(4,5)P2 synthesis through direct activation of PIPKIalpha enzyme activity.

Plasmalemmal Phosphatidylinositol-4,5-Bisphosphate Level Regulates the Releasable Vesicle Pool Size in Chromaffin Cells

During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] level in bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2 affect secretion. Membrane levels of PI(4,5)P2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2 signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase I, or infusion of PI(4,5)P2 through the patch pipette, increased the PI(4,5)P2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2 from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5)P2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2 levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2 level secretion scales with plasma membrane PI(4,5)P2 content and that PI(4,5)P2 has an early effect on secretion by regulating the number of vesicles ready for release.

Role of Ligands in Accumulation and Fractionation of Rare Earth Elements in Plants: Examples of Phosphate and Citrate

Few studies have been carried out on the effects of ligands on rare earth element (REE) bioaccumulation processes. In this study, the effects of phosphate (Pi, an inorganic ligand) and citrate (an organic ligand) on accumulation and fractionation of REEs in wheat were investigated using aqueous culture with extraneous mixed REEs (MRE). The results show that initial Pi solution culture at various levels followed by exposure to a fixed-MRE solution did not significantly change the total concentrations of REEs (SigmaREE) in roots, whereas the SigmaREE in leaves dramatically decreased with increasing levels of Pi applied. Simultaneous culture of wheat with mixture of MRE and citrate solutions caused obvious decreases of the SigmaREE in both roots and leaves. Compared with MRE, significant fractionations of REEs were found in wheat organs when no ligand was applied. Notable middle REE (MREE) enrichment and M-type tetrad effect were observed in the roots, and heavy REE (HREE) enrichment and W-type tetrad effect existed in the leaves. Pi treatments did not significantly affect the fractionations of REEs in the roots, but enrichment of HREEs in the leaves slightly increased at the highest level of Pi applied. Fractionations of REEs in both roots and leaves decreased with increasing levels of citrate applied; at higher levels of citrate (> or =150 microM), no above fractionation features were observed in wheat, but light REE (LREE) enrichment existed in the roots and leaves. The results indicate that ligands might play important roles in accumulation and fractionation of REEs during bioaccumulation processes.

Early Biochemical Alterations in Manganese Toxicity: Ameliorating Effects of Magnesium Nitrate and Vitamins

Manganese-induced early biochemical changes and effects of supplementation of magnesium nitrate (Mg(NO3)2) and antioxidant vitamins (A, C, D and E) were studied in rats intoxicated with manganese. Significant elevation in the level of chlorides in plasma, erythrocytes, liver and cerebellum, and a decrease in plasma inorganic phosphate (pi) with an increase in liver pi were observed in animals exposed to manganese as compared to controls. The level of erythrocyte-acid labile phosphate (ALP), nicotinamide adeninedinucleotide (NAD+) and plasma sialic acid (N-acetylneuraminic acid, NANA) also increased significantly. Elevated levels of chlorides in plasma, erythrocytes and cerebellum reversed to normal control values whereas liver chlorides restored partially by the supplementation of Mg(NO3)2. Vitamins supplementation was effective to reverse chlorides level in erythrocytes, liver and cerebellum. Decreased level of pi in plasma and the highly elevated level of erythrocyte ALP were also recovered in animals received Mg(NO3)2 in addition to MnSO4. However, such effect of Mg(NO3)2 was not seen in lowering the elevated level of NANA that restored by the administration of vitamins. Thus, the early alterations in plasma levels of chlorides, pi, and NANA and erythrocyte-ALP seem to be an indicative of early manganese toxicity while Mg(NO3)2 and vitamins supplementation appear to provide, at least in part, protection against manganese toxicity.