P3 Concentration Research Articles

Ethanol Triggers Neural Crest Apoptosis through the Selective Activation of a Pertussis Toxin–Sensitive G Protein and a Phospholipase Cβ–Dependent Ca2+ Transient

Alcohol is a potent neurotoxin that triggers the selective apoptosis of neuronal populations in the developing fetus. For neural crest cells, clinically relevant ethanol levels (0.3%) rapidly elicit a phospholipase C (PLC)-dependent intracellular Ca2+ transient that is sufficient to activate apoptosis. We investigated the biochemical origins of this Ca2+ transient. Three somite chick embryos (stage 8-) were pretreated with agonists and antagonists of PLC signaling pathways before ethanol challenge. The resulting intracellular Ca2+ release was quantified using Fluo-3; apoptosis was assessed using vital dyes. Pretreatment of embryos with PLC antagonists U73122 or ET-18-OCH3 confirmed that a phosphoinositide-specific PLC was required for both the ethanol-dependent Ca2+ transient and subsequent cell death. Ethanol rapidly elevated intracellular inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the rostral portion of the embryo that contains neural crest progenitors. The Ins(1,4,5)P3 receptor antagonist xestospongin C blocked the appearance of the ethanol-dependent Ca2+ transient. Pretreatment with the pan-Galpha protein antagonist GDPbetaS, but not with the tyrosine kinase antagonist genistein, suppressed ethanol's ability to elicit the Ca2+ transient, suggesting that a rise in PLC activity and Ins(1,4,5)P3 concentration originates from stimulation of heterotrimeric G proteins. To probe the identity of this G protein, embryos were treated with G protein antagonists. Pertussis toxin and NF023 suppressed the ethanol-induced Ca2+ transient and subsequent neural crest apoptosis, whereas suramin was weakly inhibitory. C3 exoenzyme was embryolethal over a wide concentration range, consistent with suggestions that Rho family GTPases participate in neural crest development. Galphai2 was identified by immunostaining in the neural crest cells. We propose a role for Galphai/o protein activation and subsequent interaction of Gbetagamma with PLCbeta in mediating the proapoptotic effects of ethanol upon the developing neural crest.

Regulation of P-Rex1 by Phosphatidylinositol (3,4,5)-Trisphosphate and Gβγ Subunits

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.

Integrated systems analysis of persistent polar pollutants in the water cycle

Persistent polar pollutants (P3) are difficult to degrade in standard waste water treatment plants. As a result, they end up in the effluent and are emitted to the surface water. In some areas, this problem is aggravated through "closed loop recycling", causing concentrations of P3 in surface water to build up over time. This could cause violation of (future) EU regulations. In the P-THREE project, various alternative waste water treatment techniques are investigated regarding their effectiveness in eliminating these substances, especially membrane bioreactor treatment and advanced oxidation processes, MBR and AOP. The integrated systems analysis which is the subject of this paper assesses these techniques in a broader systems context: (1) the life-cycle of the P3, (2) the life cycle of the WWTPs, and (3) the WWTP life cycle costs.

Complex changes in cellular inositol phosphate complement accompany transit through the cell cycle

Inositol polyphosphates other than Ins(1,4,5)P3 are involved in several aspects of cell regulation. For example, recent evidence has implicated InsP6, Ins(1,3,4,5,6)P5 and their close metabolic relatives, which are amongst the more abundant intracellular inositol polyphosphates, in chromatin organization, DNA maintenance, gene transcription, nuclear mRNA transport, membrane trafficking and control of cell proliferation. However, little is known of how the intracellular concentrations of inositol polyphosphates change through the cell cycle. Here we show that the concentrations of several inositol polyphosphates fluctuate in synchrony with the cell cycle in proliferating WRK-1 cells. InsP6, Ins(1,3,4,5,6)P5 and their metabolic relatives behave similarly: concentrations are high during G1-phase, fall to much lower levels during S-phase and rise again late in the cycle. The Ins(1,2,3)P3 concentration shows especially large fluctuations, and PP-InsP5 fluctuations are also very marked. Remarkably, Ins(1,2,3)P3 turns over fastest during S-phase, when its concentration is lowest. These results establish that several fairly abundant intracellular inositol polyphosphates, for which important biological roles are emerging, display dynamic behaviour that is synchronized with cell-cycle progression.

Expression of the bovine aromatase cytochrome P450 gene (Cyp19) is primarily regulated by promoter 2 in bovine follicles and by promoter 1.1 in corpora lutea.

Follicular development and differentiation are closely associated with increasing steroidogenesis. During the present study transcript concentration of Cyp19, Cyp11A1, and 3beta-hydroxysteroid dehydrogenase delta (3beta-HSD) encoding the steroidogenic enzymes P450(arom), P450(SCC), and 3beta-HSD were determined by real-time PCR in bovine granulosa cells (GC) as potential markers for follicular differentiation. Ovaries were collected from a local abattoir (experiment 1) and from synchronized animals at day 4 of estrus cycle (experiment 2). To study effects of luteinization, steroidogenic transcripts were also quantified in corpora lutea (CL) 4 and 20 days after fertilization. In most follicles, all three steroidogenic transcripts were detected, however, at very different concentration. Expression of 3beta-HSD and Cyp11A1 was highly significantly co-regulated and showed a significant correlation with follicular size. Contrary, Cyp19 expression was extremely variable even in follicles of similar size. Cyp19 transcripts were derived predominantly from promoter P2 and less abundant from promoters P1.1 and P1.5. After luteinization, the concentration of 3beta-HSD and Cyp11A1 transcripts increased (75-fold and fivefold, respectively) whereas the Cyp19 transcript level dropped (160-fold). Residual Cyp19 transcripts in CL were almost exclusively derived from P1.1. The data indicate that Cyp19 expression in GC is predominantly regulated by P2 and to a minor extend by P1.1, whereas P1.1 is almost exclusively responsible for residual Cyp19 expression in CL. Correlation analyses suggest that the expression of 3beta-HSD and Cyp11A1 primarily depend on the size of follicles whereas the concentration of P2 derived Cyp19 transcripts in GC is a marker for follicular differentiation towards selection and dominance.

Association and Dissociation Kinetics for CheY Interacting with the P2 Domain of CheA

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA→CheY phospho-transfer reaction is 100–1000 times faster than the His→Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His→Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA. P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs. In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA→CheY phospho-transfer by a factor of 50–100. Here we examined the kinetics of the binding interactions between CheY and P2. The rapid association reaction ( k assn ∼10 8 M −1 s −1 at 25 °C and μ=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited. Ionic strength (μ) had a moderate effect on k assn in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye–Hückel shielding. The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation. Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA. However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k cat differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK–RR pairs of slower systems.

Cytosolic Multiple Inositol Polyphosphate Phosphatase in the Regulation of Cytoplasmic Free Ca2+ Concentration

Multiple inositol polyphosphate phosphatase (MIPP) is an enzyme that, in vitro, has the interesting property of degrading higher inositol polyphosphates to the Ca2+ second messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), independently of inositol lipid breakdown. We hypothesized that a truncated cytosolic form of the largely endoplasmic reticulum-confined MIPP (cyt-MIPP) could represent an important new tool in the investigation of Ins(1,4,5)P3-dependent intracellular Ca2+ homeostasis. To optimize our ability to judge the impact of cyt-MIPP on intracellular Ca2+ concentration ([Ca2+]i) we chose a poorly responsive beta-cell line (HIT M2.2.2) with an abnormally low [Ca2+]i. Our results show for the first time in an intact mammalian cell that cyt-MIPP expression leads to a significant enhancement of Ins(1,4,5)P3 concentration. This is achieved without a significant interference from other cyt-MIPP-derived inositol phosphates. Furthermore, the low basal [Ca2+]i of these cells was raised to normal levels (35 to 115 nm) when they expressed cyt-MIPP. Noteworthy is that the normal feeble glucose-induced Ca2+ response of HIT M2.2.2 cells was enhanced dramatically by mechanisms related to this increase in basal [Ca2+]i. These data support the use of cyt-MIPP as an important tool in investigating Ins(1,4,5)P3-dependent Ca2+ homeostasis and suggest a close link between Ins(1,4,5)P3 concentration and basal [Ca2+]i, the latter being an important modulator of Ca2+ signaling in the pancreatic beta-cell.

Muscarinic receptor-mediated activation of p70 S6 kinase 1 (S6K1) in 1321N1 astrocytoma cells: permissive role of phosphoinositide 3-kinase.

In 1321N1 astrocytoma cells, carbachol stimulation of M3 muscarinic cholinergic receptors, coupled to phospholipase C, evoked a persistent 10-20-fold activation of p70 S6 kinase (S6K1). This response was abolished by chelation of cytosolic Ca2+ and reproduced by the Ca2+ ionophore ionomycin, but was not prevented by down-regulation or inhibition of protein kinase C. Carbachol-stimulated activation and phosphorylation of S6K1 at Thr389 were prevented by rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), or by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor. Carbachol also stimulated the phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a second mTOR-dependent event, with similar potency to its effect on S6K1. This response was blocked by rapamycin, but was not markedly affected by 100 nM wortmannin, implying separate roles for mTOR and PI3K in S6K1 activation. Wortmannin abolished the carbachol-stimulated rise in PtdIns(3,4,5)P3 and greatly reduced unstimulated levels of this lipid. By contrast, an inhibitor of epidermal growth factor receptor kinase, AG1478, which prevents carbachol-stimulated ErbB3 transactivation, PI3K recruitment and protein kinase B activation in 1321N1 cells, reduced activation of S6K1 by no more than 30%. This effect was overcome by 10 nM insulin, which on its own did not stimulate S6K1, but increased cellular PtdIns(3,4,5)P3 concentrations comparably with carbachol alone. These observations distinguish obligatory roles for mTOR and PI3K in regulating S6K1, but imply that minimal PI3K activity is sufficient to permit stimulation of S6K1 by other activating factors such as increased cytosolic Ca2+ concentrations, which are essential to the muscarinic receptor-mediated response. Moreover, 4E-BP1 and hence, presumably, mTOR can be regulated independently of PI3K activation through these mechanisms.

Improvement of the nutritive value of soybean meal by protease and α-galactosidase treatment in broiler cockerels and broiler chicks

1. Tube-fed broiler cockerels were used in three experiments to measure the effect of different enzyme treatments on true metabolisable energy (TME) and true nitrogen digestibility (TND) of commercial solvent-extracted, heat-treated soybean meal (SBM). 2. In experiment 1, proteases P2 and P3 (from Aspergillus niger) and α-galactosidase (from A. oryzae) improved TME and TND while protease P1 (from Bacillus subtilis) had little effect. The effects of enzyme treatment were similar whether treatment was applied by pre-incubation of enzymes (proteases P1, P2 and P3, 1·0 g/kg; α-galactosidase, 0·1 g/kg) with SBM for 2 h at 50°C or by simple mixing of enzymes (proteases P1, P2 and P3, 0·25 g/kg; α-galactosidase, 0·1 g/kg) with SBM prior to feeding. 3. In experiment 2, the effects of mixing SBM with each of protease P1 (0 or 0·25 g/kg), protease P3 (0 or 0·25 g/kg) or α-galactosidase (0 or 0·1 g/kg) alone or in all possible combinations were studied. Effects of protease P1 were limited, but protease P3 and α-galactosidase improved TME and TND. There were significant interactions between protease P3 and α-galactosidase for both TME and TND; the response when enzymes were applied together was less than that expected from application of the enzyme preparations individually. 4. In experiment 3, the effect of varying the concentration of protease P3 (0, 0·1, 0·25 and 1·0 g/kg SBM dry matter) and α-galactosidase (0, 0·025, 0·0625 and 0·25 g/kg SBM dry matter) mixed with SBM alone or in all possible combinations of protease P3 and α-galactosidase were assessed. Increases in TME and TND for both enzymes were non-linear with the greatest response occurring at the lowest concentration of each enzyme. There were also interactions between the two enzyme preparations. 5. Finally, either protease P3 (0, 0·1 and 1·0 g/kg SBM dry matter) or α-galactosidase (0, 0·025 and 0·25 g/kg SBM dry matter) were mixed with SBM alone or in all possible combinations and treated SBMs incorporated into semi-purified diets containing 450 g SBM/kg as the sole source of dietary N and offered to growing broiler chicks for 21 d. Similar patterns of increases in chick growth rate and diet digestibility to those recorded in experiment 3 were obtained when protease P3 and α-galactosidase where included in the diets. 6. It is concluded that responses measured by tube-feeding SBM treated with protease P3 and α-galactosidase were similar to responses obtained with growing broiler chicks. Further, these responses were obtained by simple addition of enzymes to diets and nutritive value of SBM was improved by both protease and α-galactosidase treatment.

Improvement of the nutritive value of soybean meal by protease and a-galactosidase treatment in broiler cockerels and broiler chicks

1. Tube-fed broiler cockerels were used in three experiments to measure the effect of different enzyme treatments on true metabolisable energy (TME) and true nitrogen digestibility (TND) of commercial solvent-extracted, heat-treated soybean meal (SBM). 2. In experiment 1, proteases P2 and P3 (from Aspergillus niger) and α-galactosidase (from A. oryzae) improved TME and TND while protease P1 (from Bacillus subtilis) had little effect. The effects of enzyme treatment were similar whether treatment was applied by pre-incubation of enzymes (proteases P1, P2 and P3, 1·0 g/kg; α-galactosidase, 0·1 g/kg) with SBM for 2 h at 50°C or by simple mixing of enzymes (proteases P1, P2 and P3, 0·25 g/kg; α-galactosidase, 0·1 g/kg) with SBM prior to feeding. 3. In experiment 2, the effects of mixing SBM with each of protease P1 (0 or 0·25 g/kg), protease P3 (0 or 0·25 g/kg) or α-galactosidase (0 or 0·1 g/kg) alone or in all possible combinations were studied. Effects of protease P1 were limited, but protease P3 and α-galactosidase improved TME and TND. There were significant interactions between protease P3 and α-galactosidase for both TME and TND; the response when enzymes were applied together was less than that expected from application of the enzyme preparations individually. 4. In experiment 3, the effect of varying the concentration of protease P3 (0, 0·1, 0·25 and 1·0 g/kg SBM dry matter) and α-galactosidase (0, 0·025, 0·0625 and 0·25 g/kg SBM dry matter) mixed with SBM alone or in all possible combinations of protease P3 and α-galactosidase were assessed. Increases in TME and TND for both enzymes were non-linear with the greatest response occurring at the lowest concentration of each enzyme. There were also interactions between the two enzyme preparations. 5. Finally, either protease P3 (0, 0·1 and 1·0 g/kg SBM dry matter) or α-galactosidase (0, 0·025 and 0·25 g/kg SBM dry matter) were mixed with SBM alone or in all possible combinations and treated SBMs incorporated into semi-purified diets containing 450 g SBM/kg as the sole source of dietary N and offered to growing broiler chicks for 21 d. Similar patterns of increases in chick growth rate and diet digestibility to those recorded in experiment 3 were obtained when protease P3 and α-galactosidase where included in the diets. 6. It is concluded that responses measured by tube-feeding SBM treated with protease P3 and α-galactosidase were similar to responses obtained with growing broiler chicks. Further, these responses were obtained by simple addition of enzymes to diets and nutritive value of SBM was improved by both protease and α-galactosidase treatment.

Modulation of inositol 1,4,5-triphosphate concentration by prolyl endopeptidase inhibition.

Prolyl endopeptidase (PEP) is a proline-specific oligopeptidase with a reported effect on learning and memory in different rat model systems. Using the astroglioma cell line U343, PEP expression was reduced by an antisense technique. Measuring different second-messenger concentrations revealed an inverse correlation between inositol 1,4,5-triphosphate [Ins(1,4,5)P3] concentration and PEP expression in the generated antisense cell lines. However, no effect on cAMP generation was observed. In addition, complete suppression of PEP activity by the specific inhibitor, Fmoc-Ala-Pyrr-CN (5 micro m) induced in U343 and other cell lines an enhanced, but delayed, increase in Ins(1,4,5)P3 concentration. This indicates that the proteolytic activity of PEP is responsible for the observed effect. Furthermore, the reduced PEP activity was found to amplify Substance P-mediated stimulation of Ins(1,4,5)P3. The effect of reduced PEP activity on second-messenger concentration indicates a novel intracellular function of this peptidase, which may have an impact on the reported cognitive enhancements due to PEP inhibition.

Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteins: an essential role for polyphosphoinositides in vivo.

Ezrin/radixin/moesin (ERM) proteins crosslink actin filaments to plasma membranes and are involved in the organization of the cortical cytoskeleton, especially in the formation of microvilli. ERM proteins are reported to be activated as crosslinkers in a Rho-dependent manner and are stabilized when phosphorylated at their C-terminal threonine residue to create C-terminal threonine-phosphorylated ERM proteins (CPERMs). Using a CPERM-specific mAb, we have shown, in vivo, that treatment with C3 transferase (a Rho inactivator) or staurosporine (a protein kinase inhibitor) leads to the dephosphorylation of CPERMs, the translocation of ERM proteins from plasma membranes to the cytoplasm and microvillar breakdown. We further elucidated that ERM protein activation does not require C-terminal phosphorylation in A431 cells stimulated with epidermal growth factor. In certain types of kidney-derived cells such as MDCK cells, however, ERM proteins appear to be activated in the absence of Rho activation and remain active without C-terminal phosphorylation. Interestingly, microinjection of an aminoglycoside antibiotic, neomycin, which binds to polyphosphoinositides, such as phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)], affected the activation of ERM proteins regardless of cell type. These findings not only indicate the existence of a Rho-independent activation mechanism of ERM proteins but also suggest that both Rho-dependent and -independent activation of ERM proteins require a local elevation of PtdIns(4,5)P(2) concentration in vivo.

Three different calcium wave pacemakers in ascidian eggs

Calcium wave pacemakers in fertilized eggs of ascidians and mouse are associated with accumulations of cortical endoplasmic reticulum in the vegetal hemisphere. In ascidians, two distinct pacemakers (PM1 and PM2) generate two series of calcium waves necessary to drive meiosis I and II. Pacemaker PM2 is stably localized in a cortical ER accumulation situated in the vegetal contraction pole. We now find that pacemaker PM1 is situated in a cortical ER-rich domain that forms around the sperm aster and moves with it during the calcium-dependant cortical contraction triggered by the fertilizing sperm. Global elevations of inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) levels produced by caged Ins(1,4,5)P3 or caged glycero-myo-PtdIns(4,5)P2 photolysis reveal that the cortex of the animal hemisphere, also rich in ER-clusters, is the cellular region most sensitive to Ins(1,4,5)P3 and acts as a third type of pacemaker (PM3). Surprisingly, the artificial pacemaker PM3 predominates over the natural pacemaker PM2, located at the opposite pole. Microtubule depolymerization does not alter the activity nor the location of the three pacemakers. By contrast, blocking the acto-myosin driven cortical contraction with cytochalasin B prevents PM1 migration and inhibits PM2 activity. PM3, however, is insensitive to cytochalasin B. Our experiments suggest that the three distinct calcium wave pacemakers are probably regulated by different spatiotemporal variations in Ins(1,4,5)P3 concentration. In particular, the activity of the natural calcium wave pacemakers PM1 and PM2 depends on the apposition of a cortical ER-rich domain to a source of Ins(1,4,5)P3 production in the cortex. Movies available on-line

The Role of Zinc Binding in the Biological Activity of Botulinum Toxin

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.

Study of the effects of vacuuming on the concentration of dust mite antigen and endotoxin.

Dust mite antigens are major sources of allergens in house dust and together with endotoxin, a proinflammatory component of gram negative bacteria also found in house dust, are important causes of tissue injury involved in the pathogenesis and severity of allergic diseases, eg, asthma. To determine the effectiveness of vacuuming in reducing the quantity of dust mite antigens, Dermatophagoides pteronyssinus (Der p and Der p2) and endotoxin using a quantitative ELISA assay and to correlate results with those obtained using a qualitative rapid dipstick method for Der p2. Four specimens of house dust were collected using a Kirby Model G5 vacuum cleaner with a Micron Magic Filtration system from an approximately 54" x 18" standardized area of rug from each of 20 homes at 4 time intervals over a 6-week period, ie, a baseline specimen #1 at 0 week; specimen #2 at 1 week; specimen #3 at 5 weeks (1 month after specimen #2); specimen #4 at 6 weeks (1 week after specimen #3). Three intervals were compared, ie, period 1-2 (1 week), period 2-3 (1 month), and period 3 to 4 (1 week). The concentrations of Der p1 and Der p2 were determined in dust samples using a standard ELISA assay and the concentration of endotoxin was detected using a limulus amebocyte lysate assay. Concentrations of Der p2, determined by the standard ELISA assay, were compared to those in the same samples determined by a rapid dipstick method. A wide range of values for total weight of unprocessed dust (0.3 to 59 g, X = 8.7) and finely sieved dust (0.1 to 19 g, X = 3) from all specimens were found. In finely sieved dust specimens the mean concentrations of Der p1, Der p2 and endotoxin were 775, 1310, and 3836 ng/g of dust, respectively. Following weekly vacuuming there was an increase in concentration of Der p1, Der p2, and endotoxin in 20%, 35%, and 63% of the houses, respectively, compared to in monthly vacuuming in which increases were seen in 65%, 50%, and 63% of the houses, respectively. In contrast, there was a decrease in concentration of Der p1, Der p2 and endotoxin with weekly vacuuming in 43%, 60%, and 37% of the houses respectively versus in monthly vacuuming in 15%, 35%, and 37% of the houses respectively. A correlation coefficient of 0.7 was found for the concentration of Der p2 in 37/40 samples tested detected using the ELISA method compared with rapid dipstick assay. The results of this study support the effectiveness of vacuuming on the reduction of dust mite antigens (Der p1 and Der p2) ie, Der p2 > Der p1. This reduction was more pronounced with weekly compared with monthly vacuuming. No reduction in the concentration of endotoxin was found. A good correlation was found between results obtained by ELISA and rapid dipstick assay for Der p2.

Sequence Analyses in PDPS/PDMS Statistical Random Copolymers

AbstractThe anionic copolymerization of hexaphenylcyclotrisiloxane (P3) and hexamethylcyclotrisiloxane (D3) under non‐equilibrium conditions was carried out for synthesizing a series of poly(diphenylsiloxane‐stat‐dimethylsiloxane) copolymers. The sequence analyses of these copolymers were studied. Sequence distribution up to the pentad level is observable in the 1H NMR spectrum. The sequence distributions calculated from a pentad model by using Bovey's method, interpret the 1H NMR spectra (SiCH3 only) of all the copolymers very well. However, the triad model was only valid for relatively lower P3 content (< 50 mol%) copolymers.

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with spontaneous calcium oscillations and enhanced calcium responses following endothelin-1 stimulation

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P4) and thereby regulates cellular transformation. To investigate the role Ins(1,4,5)P3-mediated Ca2+ oscillations play in cellular transformation, we studied Ins(1,4,5)P3-mediated Ca2+ responses in cells underexpressing the 43 kDa 5-phosphatase. Chronic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fold increase in the resting Ins(1,4,5)P3 concentration and a 4.1-fold increase in basal intracellular Ca2+. The increased Ins(1,4,5)P3 levels resulted in partial emptying (40%) of the Ins(1,4,5)P3-sensitive Ca2+ store, however, store-operated Ca2+ influx remained unchanged. In addition, Ins(1,4,5)P3 receptors were chronically down-regulated in unstimulated cells, as shown by a 53% reduction in [3H]Ins(1,4,5)P3 binding to microsomal receptor sites. Agonist stimulation with endothelin-1 resulted in the rapid rise and fall of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels, with no significant differences in the rates of hydrolysis of these second messengers in antisense- or vector-transfected cells. These studies indicate, in contrast to its predicted action, the 43 kDa 5-phosphatase does not metabolise Ins(1, 4,5)P3 and Ins(1,3,4,5)P4 post agonist stimulation. Cells with decreased 43 kDa 5-phosphatase activity exhibited spontaneous Ca2+ oscillations in the absence of any agonist stimulation, and increased sensitivity and amplitude of intracellular Ca2+ responses to both high and low dose endothelin-1 stimulation. We conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1,4, 5)P3-induced Ca2+ spiking, both in the unstimulated cell and following agonist stimulation. We propose the enhanced Ca2+ oscillations may mediate cellular transformation in cells underexpressing the 43 kDa 5-phosphatase.

Preconditioning-induced cardioprotection and release of the second messenger inositol (1,4,5)-trisphosphate are both abolished by neomycin in rabbit heart

The mechanisms responsible for infarct size reduction with preconditioning remain controversial. Our aim was to determine whether release of the second messenger inositol (1,4,5)-triphosphate (Ins(1,4,5)P3) during the preconditioning stimulus may play a role. To test this concept, Langendorff-perfused rabbit hearts underwent sham perfusion, 5 min of coronary artery occlusion (CO), or 5 min of CO + infusion of neomycin, an agent which inhibits formation of Ins(1,4,5)P3. Direct quantitation (by competitive binding assay) revealed a 2-fold increase in Ins(1,4,5)P3 content with brief ischemia vs shams (0.69 +/- 0.14 vs 0.34 +/- 0.05 pmol/mg tissue; p < .05) that was blocked by neomycin (0.15 +/- 0.04 pmol/mg). Infarct size (by tetrazolium staining) was assessed in additional hearts that underwent 30 min of sustained CO and 2 h of reperfusion. As expected, two 5-min episodes of preconditioning ischemia reduced infarct size versus controls (30 +/- 6% versus 63 +/- 3% of the myocardium at risk; p < .01). In contrast, infarct size was comparable (54-56% of the risk region) in neomycin-treated control and preconditioned hearts. These results demonstrate that myocardial Ins(1,4,5)P3 content is increased in response to brief preconditioning ischemia and are consistent with the concept that Ins(1,4,5)P3 may be a potential mediator of infarct size reduction with preconditioning in isolated rabbit heart.

Light Induces lnositol Trisphosphate Elevation in Blepharisma japonicum.

Abstract- Photoinduced formation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3 ) was examined using a specific radioimmu-noassay to investigate the molecular mechanisms of light signal transduction mediating photophobic responses in the ciliate Blepharisma japonicum. Application of light stimuli of moderate intensity to dark-adapted cells induced a rapid and significant increase in the basal level of Ins (1,4,5)P3 , with a peak at about 20 s. Thereafter, the level of Ins (1,4,5)P3 declined to the resting value within the subsequent 100 s. Light stimuli of higher intensity raised the cell Ins (1,4,5)P3 content to still higher levels within about 20 s, but the decaying time course was considerably prolonged. In ciliates incubated under dark conditions with agents interfering with the inositol signalling pathway, like neomycin and Li+ the basal levels of Ins (1,4,5)P3 were lower than in control cells. A photoinduced rise of Ins (1,4,5)P3 , content in ciliates treated with neomycin or Li+ was significantly inhibited in a dose-dependent manner. Depolarizing ionic stimuli in dark-adapted ciliates induced no significant alterations of the resting Ins (1,4,5)P3 level, indicating a lack of a contribution of this kind of stimulation to the inositol turnover. These studies are the first in vivo demonstration of a possible role for inositol trisphosphate as a second messenger in the light signal transduction process in the ciliate B. japonicum.

Acute desensitization of phospholipase C-coupled muscarinic M3 receptors but not gonadotropin-releasing hormone receptors co-expressed in alphaT3-1 cells: implications for mechanisms of rapid desensitization.

In the present study we have expressed the muscarinic M3 receptor in an immortalized mouse pituitary cell line (alphaT3-1), which expresses an endogenous gonadotropin-releasing hormone (GnRH) receptor, to examine potential differences in acute receptor regulation. Both of these receptors couple to the activation of phosphoinositide-specific phospholipase C (PLC) in these cells and we demonstrate that, despite expression in the same cell background, acute desensitization is a feature of muscarinic M3 receptors but not of GnRH receptors. We show that, when the concentrations of GnRH and methacholine are matched to give approximately equivalent maximal elevations of Ins(1,4,5)P3, the GnRH receptor is able to sustain PLC activity at the initial rate, whereas the muscarinic M3 receptor cannot. Thus PLC-activating G-protein-coupled receptors are able to undergo rapid desensitization in this cell line, indicating that the desensitization profile is receptor-specific rather than cell-specific. This argues strongly that post-receptor regulatory features do not have a prominent role in mediating rapid desensitization in these cells. Furthermore GnRH receptor-mediated PLC activity is sustained despite a marked and persistent depletion in the steady-state level of PtdIns(4,5)P2. In contrast, activation of muscarinic receptors is not sustained despite only a transient decrease in PtdIns(4,5)P2 concentration. Thus, whereas the contribution of PtdIns(4,5)P2 depletion to the temporal profile of receptor-mediated PLC signalling has been difficult to assess, the present results demonstrate that this is unlikely to be of importance in these cells. We suggest that unique structural features of the GnRH receptor result in a lack of appropriate regulatory phospho-acceptor sites and that the absence of agonist-dependent phosphorylation might underlie the lack of acute regulation.