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Studies have examined the function of PI 3-kinase in the early developmental processes that operate in oocytes or early embryos of various species. However, the roles of egg-associated PI 3-kinase and Akt, especially in signal transduction at fertilization, are not well understood.
Here we show that in Xenopus eggs, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), LY294002 inhibits sperm-induced activation of the tyrosine kinase Src and a transient increase in the intracellular concentration of Ca2+ at fertilization. LY294002 also inhibits sperm-induced dephosphorylation of mitogen-activated protein kinase, breakdown of cyclin B2 and Mos, and first embryonic cleavage, all of which are events of Ca2+-dependent egg activation. In fertilized eggs, an 85-kDa subunit of PI 3-kinase (p85) undergoes a transient translocation to the low-density, detergent-insoluble membranes (membrane microdomains) where Src tyrosine kinase signaling is operating. However, the tyrosine phosphorylation of p85 in fertilized eggs is not as evident as that in H2O2-activated eggs, arguing against the possibility that PI 3-kinase is activated by Src phosphorylation. Nevertheless, sperm-induced activation of PI 3-kinase has been demonstrated by the finding that Akt, a serine/threonine-specific protein kinase, is phosphorylated at threonine-308. The threonine-phosphorylated Akt also localizes to the membrane microdomains of fertilized eggs. Application of bp(V), an inhibitor of PTEN that dephosphorylates PIP3, the enzymatic product of PI 3-kinase, promotes parthenogenetic activation of Xenopus eggs. In vitro kinase assays demonstrate that PIP3 activates Src in a dose-dependent manner.
These results suggest that PI 3-kinase is involved in sperm-induced egg activation via production of PIP3 that would act as a positive regulator of the Src signaling pathway in Xenopus fertilization.
At fertilization, the union of egg and sperm promotes a series of biochemical and cell biological changes within the fertilized egg. This phenomenon is termed 'egg activation' [1-3]. A trigger of egg activation, which acts inside the fertilized egg after the egg-sperm union, is a transient increase in intracellular Ca2+ (Ca2+ transient) [4-6]. One important consequence of egg activation is that the egg acquires the ability to exclude additional fertilizing sperm (block to polyspermy). In many, but not all species, the block to polyspermy is achieved by an altered membrane potential and/or by the formation of a fertilization envelope. Another important consequence is that the activated egg resumes meiotic cell division. In the case of amphibian and most mammalian species, the meiotic cell cycle of unfertilized eggs pauses at metaphase II, and successful fertilization promotes meiotic resumption and extrusion of the second polar body. These egg activation events are followed by the fusion of maternal and paternal nuclei and the initiation of embryonic cell division that produce an offspring.
The sperm-induced Ca2+ transient, a key event in the initiation of egg activation, is commonly mediated by inositol 1,4,5-trisphosphate (IP3), a second messenger that is produced by the phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate. The molecular mechanism operating between egg-sperm membrane interaction/fusion and the activation of PLC, however, varies among species: in mammals and the newt Cynops pyrrohogaster, introduction of the sperm-derived proteins PLCζ [7] and citrate synthase [8], respectively, may account for this task. In these cases, egg-sperm membrane fusion, rather than egg-sperm membrane interaction, is crucial for initiating the Ca2+ transient. On the other hand, for some sea invertebrates, fish and frogs, there is still a debate over the mechanism by which the egg undergoes a Ca2+ transient. That sequential activation of the egg-associated Src tyrosine kinase and PLCγ is required for the Ca2+ transient in the sea urchin, starfish, fish, and frog [9-14] suggests that these species employ the membrane interaction machinery. Also, some membrane-associated molecules have been postulated as sperm-interacting and signal-transducing elements in Xenopus eggs [15-18].
Several studies have evaluated the function of PI 3-kinase in the early developmental processes that operate in oocytes or early embryos of various species. In Xenopus, PI 3-kinase and Akt are required for insulin-induced, but not progesterone-induced, oocyte maturation [19,20], although one report has shown a requirement of PI 3-kinase for progesterone-induced oocyte maturation [21]. There are also reports that the activation of γ-subspecies of PI 3-kinase [22] or application of wortmannin [23] induces oocyte maturation. On the other hand, oocyte maturation in the ascidian [24], mouse [25,26] and starfish [27] has been shown to require activity of PI 3-kinase. Oocyte-specific deletion of PTEN is shown to cause premature activation of the primordial follicle cells [28], suggesting that a precise level of PIP3 is important for this process.
Moreover, the importance of PI 3-kinase and/or Akt has been demonstrated in FGF-dependent signal transduction [29,30] and glucose transport in Xenopus oocytes [31], the first mitotic cell division in the sea urchin [32] and starfish [33], autocrine-mediated survival signaling of mouse two-cell embryos [34], mesoderm induction [35], gastrulation [36,37] and neurogenesis [38] in Xenopus. Collectively, these studies demonstrate the general importance of PI 3-kinase and its enzymatic products in several aspects of development. However, a study on egg-associated PI 3-kinase and Akt with a focus on fertilization signaling has yet to be done, though Mehlmann et al. [39] found that LY294002 does not inhibit Ca2+ transients in fertilized mouse eggs.
Here, we provide evidence that the sperm-induced Ca2+ transient requires the activity of the egg-associated PI 3-kinase in Xenopus. Many somatic cell systems utilize PI 3-kinase as a downstream effector of tyrosine kinase signaling [40,41]. In fertilized Xenopus eggs, however, tyrosine phosphorylation of the p85 subunit of PI 3-kinase is not evident. On the other hand, PI 3-kinase seems to act as an upstream, positive regulator for the Src-PLCγ pathway, because the PI 3-kinase inhibitor LY294002 inhibits the sperm-induced activation of Src, Ca2+ transient, and subsequent egg activation events. Subcellular fractionation studies demonstrate that the 85-kDa subunit of PI 3-kinase undergoes a transient translocation to low-density, detergent-insoluble membranes (membrane microdomains), where Src is present even before fertilization, awaiting a fertilization signal from sperm [42]. Activation of PI 3-kinase in fertilized Xenopus eggs is suggested by the finding that Akt becomes phosphorylated at threonine-308 in fertilized eggs. These results highlight for the first time that activity of the egg PI 3-kinase may play an important role in signal transduction for fertilization.
We first examined the effect of LY294002, a potent PI 3-kinase inhibitor, on sperm-induced activation of the Src-PLCγ pathway. We microinjected LY294002 into unfertilized Xenopus eggs at a final concentration of 10 μM. The injected eggs were inseminated for 5 min, fractionated, and examined for sperm-induced activation and phosphorylation of Src and PLCγ, using immunoprecipitation, immunoblotting, and/or in vitro kinase assays (IVKA). LY294002 inhibited the sperm-induced activation of Src, as judged by the phosphorylation of tyrosine-419 in Src (Figure 1A, IB: phospho-Src, lanes 1, 2, and 6) and by the kinase activity toward a synthetic peptide substrate (Figure 1A, IVKA: phospho-Cdk1). Tyrosine phosphorylation of PLCγ was also inhibited by LY294002 (Figure 1A, IB: phospho-PLCγ). As we reported earlier, a Src-specific inhibitor PP2 also had an inhibitory effect (Figure 1A, lane 3). Neither the inactive analog PP3 (lane 4) nor U73122 (lane 5), a PLC-specific inhibitor, showed such an effect.
Figure 1. Inhibition of early events of Xenopus egg fertilization by LY294002, a potent PI 3-kinase inhibitor. (A) Xenopus unfertilized eggs were microinjected with DMSO alone (lanes 1 and 2), 10 μM PP2 (lane 3), 10 μM PP3 (lane 4), 10 μM U73122 (lane 5), or 10 μM LY294002 (lane 6), and subjected to no treatment (lane 1, - sperm) or insemination for 5 min (lanes 2-6, + sperm). SDS (0.1%)-solubilized egg membrane fractions (500 μg/lane) abundant in Src and PLCγ were analyzed for tyrosine phosphorylation of Src (top panel, IB: phospho-Src), activation of Src (middle panels, IVKA: phospho-Cdk1; IVKA: protein amounts of the immunoprecipitated Src), and tyrosine phosphorylation of PLCγ (bottom panel, IB: phospho-PLCγ) as described in "Methods". Asterisks in each panel indicate the positions of the protein bands of interest. (B) Shown are representative traces of sperm-induced Ca2+ release, as monitored by the fluorescent ratio signal, in Xenopus eggs that were co-injected with fura-2 and LY294002 (0 or 10 μM) and inseminated as described in "Methods". (C) Triton X-100-solubilized extracts (100 μg/lane) were prepared from Xenopus eggs that were injected with the indicated inhibitors (lanes 3-5, each 10 μM) and inseminated for 40 min. Extracts from DMSO (0.2%)-injected unfertilized (lane 1) or fertilized eggs (lane 2) were also prepared as controls. Samples were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against phospho-MAPK (top panel), cyclin B2 (middle panel), or Mos (bottom panel) as described in "Methods". (D) The occurrence of first embryonic cleavage was evaluated in fertilized Xenopus embryos that were injected with the indicated concentrations of LY294002 and inseminated for 100 min. Values indicated on each bar are the number of cleaved embryos per number of embryos tested.
We have previously shown that activation of Src and PLCγ is necessary for sperm-induced activation of Xenopus eggs. Therefore, we examined the effect of LY294002 on the downstream events of the Src-PLCγ pathway; namely the transient Ca2+ release (Figure 1B), inactivation of components of the cytostatic factor (Figure 1C), and first cell cleavage (Figure 1D). All of the LY294002-injected eggs exhibited features of an impaired transient Ca2+ release, as monitored using the fluorescent signal of fura-2 that was introduced into the eggs (Figure 1B). Seven of nine eggs tested showed no transient Ca2+ release, and two of nine showed a transient Ca2+ release of decreased amplitude (Figure 1B). LY294002 also had an inhibitory effect on the Ca2+-dependent breakdown of the Mos protein kinase and cyclin B2 as well as dephosphorylation of mitogen-activated protein kinase (MAPK) (Figure 1C). These Ca2+-dependent events are required for cancelling the arrest of the second meiotic cell cycle and for starting embryonic cell division. Actually, eggs injected with 10 μM LY294002 failed to undergo normal first cell cleavage (Figure 1D).
We also examined the effect of LY294002 on egg activation, as evaluated by the occurrence of cortical contraction, induced by several artificial egg activators. LY294002 effectively inhibited the egg activation induced by cathepsin B at 5 U/ml (23 of 90 eggs showed cortical contraction). On the other hand, the egg activation induced by A23187 at 2 μM and that by H2O2 at 10 mM were not inhibited; 85 of 90 eggs and 76 of 90 eggs were activated, respectively. These data suggest that egg activation by cathepsin B, which mimics the action of sperm protease and requires the egg Src activity, also requires the activity of PI 3-kinase. It should be noted that we did not employ LY294002 higher than 10 μM, because it required more volume of DMSO solution to be injected that would show a toxic effect on eggs.
Immunoblotting demonstrated that an 85-kDa subunit of PI 3-kinase, hereafter termed p85; is expressed in unfertilized Xenopus eggs (Figure 2A). Subcellular fractionation experiments demonstrated that p85 is predominantly present in the detergent-soluble fractions of unfertilized eggs (Figure 2B). However, fertilization promoted a transient accumulation of p85 in low-density, detergent-insoluble membranes (LD-DIM or membrane microdomains) (Figure 2C, IB: p85). The sperm-induced translocation of p85 to membrane microdomains (2 min) occurred as early as the transient translocation of PLCγ (Figure 2C, IB: PLCγ, 2 min) and preceded the tyrosine phosphorylation of uroplakin III (Figure 2C, IB: pUPIII, 5 min), both of which are biochemical signs of the activation of Src in fertilized Xenopus eggs. These translocation and phosphorylation events were shown to cease in 40-min inseminated egg samples, where dephosphorylation of MAPK is taking place (Figure 2C, IB: pMAPK, 40 min). Translocation of p85 to the egg membrane microdomains was also seen when eggs were treated with either hydrogen peroxide (H2O2) or cathepsin B, which are artificial egg activators involved in the activation of Src (Figure 2B, IB: p85, lanes 4 and 5). On the other hand, translocation of p85 did not occur when eggs were activated by the Ca2+ ionophore A23187 (Figure 2B, IB: p85, lane 3). Under the same experimental conditions, we evaluated the tyrosine phosphorylation of p85 by immunoblotting with anti-phosphotyrosine antibody. It was shown that only the H2O2 treatment of eggs promoted an increase in the tyrosine phosphorylation of p85 (Figure 2B, IB: phospho-p85, lanes 4 and 9). These results suggest that any sperm-induced activation of PI 3-kinase involves the transient translocation of p85 to membrane microdomains, but not increased tyrosine phosphorylation of p85.
Figure 2. Subcellular localization and tyrosine phosphorylation of an 85-kDa subunit of PI 3-kinase before and after fertilization of Xenopus eggs. (A) Triton X-100-solubilized extracts of Xenopus unfertilized eggs (lane 1, 100 μg/lane) and 293 human embryonic kidney cells (lane 2, 20 μg/lane) were separated by SDS-PAGE and analyzed by immunoblotting with the anti-p85 subunit of PI 3-kinase antibody. An asterisk indicates the position of the immunoreactive 85-kDa protein (doublets in the egg extracts). Molecular size markers (in kDa) used are also indicated. (B) Low density, detergent-insoluble membrane (LD-DIM) (left panels) and detergent-soluble (right panels) fractions were prepared from Xenopus eggs that had been untreated (lanes 1 and 6) or activated by 106/ml sperm (lanes 2 and 7), 0.5 μM A23187 (lanes 3 and 8), 10 mM H2O2 (lanes 4 and 9), or 5 U/ml cathepsin B (lanes 5 and 10). Protein samples (LD-DIM fractions, 5 μg/lane; detergent-soluble fractions, 800 μg/lane) were immunoprecipitated with the anti-p85 subunit of PI 3-kinase antibody and the immunoprecipitates were analyzed by immunoblotting with the same antibody (IB: p85) or with the anti-phosphotyrosine antibody (IB: phospho-p85). Asterisks indicate the positions of the p85 bands in each panel. (C) LD-DIM fractions were prepared from Xenopus eggs that had been inseminated for the periods indicated (0-40 min). Protein samples (5 μg/lane) were analyzed for the presence of PLCγ, p85 subunit of PI 3-kinase, or tyrosine-phosphorylated UPIII (pUPIII) by immunoprecipitation and immunoblotting (IB) as described in "Methods". Triton X-100-solubilized extracts of Xenopus unfertilized eggs (100 μg/lane) were also analyzed for the presence of phosphorylated MAPK (pMAPK) by direct immunoblotting as in Figure 1C. Asterisks indicate the positions of the protein bands of interest.
We next examined whether the activity of PI 3-kinase is upregulated in fertilized Xenopus eggs. To this end, we evaluated the phosphorylation status of Akt, a serine/threonine-specific protein kinase known to be a downstream target of the activated PI 3-kinase. Immunoblotting demonstrated that a 60-kDa Akt protein is present in unfertilized Xenopus eggs (Figure 3A, IB: Akt). Fertilization promoted phosphorylation of Akt at threonine-308, one of the activating phosphorylation sites in Akt (Figure 3B, IB: pThr308-Akt). On the other hand, serine-473, another activating phosphorylation site of Akt, remained unphosphorylated in both unfertilized and fertilized eggs (Figure 3B, IB: pSer473-Akt). LY294002, but not PP2, blocked the threonine phosphorylation of Akt (Figure 3C, IB: pThr308-Akt). These results suggest that sperm-induced phosphorylation of Akt at threonine-308 requires the activity of PI 3-kinase, but not the activity of Src tyrosine kinase.
Figure 3. Phosphorylation and translocation of a serine/threonine-specific protein kinase Akt in fertilized Xenopus eggs. (A) Triton X-100-solubilized extracts of Xenopus unfertilized eggs (lane 1, 100 μg/lane) and 293 human embryonic kidney cells (lane 2, 20 μg/lane) were separated by SDS-PAGE and analyzed by immunoblotting with the anti-mammalian Akt antibody. Molecular size markers (in kDa) used are also indicated. (B) Xenopus unfertilized eggs were microinjected with DMSO alone, 10 μM PP2, or 10 μM LY294002, and subjected to no treatment (0 min) or insemination for the periods indicated (1-20 min). Triton X-100-solubilized extracts (100 μg/lane) were prepared and analyzed by immunoblotting with the anti-Akt antibody (IB: Akt), anti-phosphorylated serine-473-specific antibody (IB: pSer473-Akt), or anti-phosphorylated threonine-308-specific antibody (IB: pThr308-Akt). (C) Effect of microinjection of PP2 (lane 3), LY294002 (lane 4), or PP3 (lane 5) on sperm-induced phosphorylation of Akt was evaluated using unfertilized (UF, lane 1) and 5-min inseminated egg samples (F5, lanes 2-5) by immunoblotting as in panel B. (D) Xenopus unfertilized eggs (UF) and 5-min inseminated eggs (F5) were subjected to subcellular fractionation by discontinuous sucrose density gradient ultracentrifugation as described in "Methods". The twelve fractions obtained were analyzed by immunoblotting with anti-Akt antibody (IB: Akt). The positions of LD-DIM fractions (fractions 4-6, 10-time concentrated) and detergent-soluble, non-microdomain fractions (fractions 10-12) are indicated. (E) LD-DIM fractions and soluble, non-microdomain fractions were prepared from unfertilized eggs (UF) and fertilized eggs (1-20 min after insemination, F1-F20). Protein samples of the same egg-equivalent amounts (about 10 eggs per lane) were analyzed for the presence of Akt by immunoprecipitation and immunoblotting with the anti-Akt antibody (IP: Akt, IB: Akt). (F) Detergent-soluble, non-microdomain fractions (20 eggs per lane) and LD-DIM fractions (2 eggs per lane) that had been prepared from unfertilized eggs (UF) and 5-min inseminated eggs (F5) were analyzed by immunoblotting with either the anti-Akt antibody (IB: Akt) or anti-phosphorylated threonine-308-specific antibody (IB: pThr308-Akt). (G) Effect of LY or PP2 on the sperm-induced translocation of the p85 subunit of PI 3-kinase (upper panel) and Akt (lower panel) was evaluated by immunoblotting of LD-DIM fractions that had been prepared from unfertilized eggs (UF) or 5-min fertilized eggs (F5). In all panels, asterisks indicate the positions of Akt or p85.
Subcellular fractionation experiments demonstrated that Akt protein undergoes translocation to membrane microdomains in fertilized eggs (Figure 3D). The translocation of Akt occurred as early as 2 min after insemination (Figure 3E), a time course that is similar to translocation of p85 (Figure 2B). Quantitative analysis demonstrated that only a small fraction of Akt localized to membrane microdomains in fertilized eggs (Figure 3F, IB: Akt). However, the threonine-308-phosphorylated forms of Akt were predominantly present in the membrane microdomains (Figure 3F, IB: pThr308-Akt). These results suggest that the sperm-induced functional interaction, and activation, of PI 3-kinase and Akt take place specifically in the egg membrane microdomains. We also examined whether the sperm-induced re-localization of Akt requires the activity of PI 3-kinase. LY294002 was shown to inhibit the sperm-induced appearance of both p85 and Akt in the membrane microdomains (Figure 3G). Like in the case of the threonine-308-phosphorylation described above, PP2 did not affect the re-localization of p85 or Akt (Figure 3G).
To examine further the involvement of the activity of PI 3-kinase in sperm-induced egg activation, we employed bp(V) [dipotassium bisperoxo (picolinato) oxovanadate (V)], a potent inhibitor of the phosphoinositide phosphatase PTEN that converts PIP3, an enzymatic product of PI 3-kinase, into PIP2. As shown in Figure 4A and 4B, bp(V) treatment of unfertilized Xenopus eggs induced a cortical contraction, a cytological event of Ca2+-dependent egg activation. Importantly, the cortical contraction induced by bp(V) was inhibited by pre-injection of LY294002 (Figure 4B). Moreover, bp(V) treatment of eggs also promoted the tyrosine phosphorylation of Src (Figure 4C). These results demonstrate that artificially elevating the PIP3 level in unfertilized eggs can promote an egg activation-like event as seen in fertilized eggs. Finally, we examined the effect of PIP3 on Src tyrosine kinase activity. An in vitro kinase assay with purified Xenopus Src demonstrated that PIP3 was capable of activating the autophosphorylation of Src (Figure 4D). Inositol phospholipids other than PIP3; namely PI, PIP, and PIP2, did not show such a Src-activating property (Figure 4D). These results indicate PIP3 to be a specific and direct activator for Src tyrosine kinase in Xenopus eggs.
Figure 4. Activation of Xenopus unfertilized eggs by bp(V), a potent inhibitor of PTEN. (A) Shown are representative photographs of Xenopus eggs before and after 20-min treatment with 200 μM bp(V). (B) The occurrence of cortical contraction as a function of time was monitored in Xenopus eggs that had been treated with sperm alone (sperm, open squares), 200 μM bp(V) alone (bp(V), open circles), sperm plus pre-injected 10 μM LY294002 (sperm + LY, closed squares), or 200 μM bp(V) plus pre-injected 10 μM LY294002 (bp(V) + LY, closed circles). (C) Xenopus eggs that had been treated with or without 200 μM bp(V) and 10 μM LY294002 for 30 min were subjected to extraction of the membrane fractions. The SDS-solubilized membrane extracts (500 μg per lane) were analyzed for tyrosine phosphorylation of Src as in Figure 1A. (D) Purified Src was pretreated with several phospholipids at the indicated concentrations (0-100 μM) and subjected to in vitro kinase assays as described in "Methods". Autophosphorylation of Src was analyzed by immunoblotting of the reaction mixtures with the phosphorylated tyrosine-419-specific antibody. Shown on the left are representative immunoblotting data. Results shown in the right are the mean ± s.e.m. of four independent experiments (at 100 μM of each phospholipids). *P < 0.01 compared with levels in the control. Src activity in the absence of phospholipids was taken as 100%.
In Xenopus eggs, the sperm-induced activation of Src tyrosine kinase plays an essential role in Ca2+ transient and egg activation [11,43,44]. The present study has indicated the activity of PI 3-kinase to connect egg-sperm interaction/fusion with the activation of Src, because 1) LY294002 inhibits the sperm-induced activation of Src, suggesting that PI 3-kinase acts as an upstream activator for Src, and 2) tyrosine phosphorylation of the p85 subunit of PI 3-kinase, a well-known phenomenon in several cellular systems, in which one or more tyrosine kinase is activated, is not stimulated in fertilized eggs. If so, what is the mechanism for Src activation by PI 3-kinase activity? One possibility is, as demonstrated in the present study, the kinase-activating interaction of PIP3 with Src. The Src homology 2 (SH2) domain of the Src protein is capable of binding PIP3 [45], supporting the possibility that interaction with PIP3 would displace the negative-regulatory intramolecular interaction involving the SH2 domain, leading to the Src kinase's activation.
In our experimental system, Wortmannin, another potent inhibitor for PI 3-kinase, is not inhibitory to the sperm-induced egg activation in Xenopus: in eggs injected with Wortmannin at up to 20 μM, sperm could promote Src activation, p85 translocation, cortical contraction, and most importantly Akt phosphorylation, all of which are events of egg activation (data not shown). There is a possibility that, in Xenopus eggs, Wortmannin binds preferentially to molecules other than PI 3-kinase. This idea is supported by the finding of Carnero and Lacal [23] that wortmannin is able to induce meiotic maturation in Xenopus oocytes at doses slightly higher that those required for complete inhibition of PI 3-kinase. The authors have also suggested that this effect is independent of the ability of Wortmannin to inhibit PI 3-kinase since LY294002 was unable to induce oocyte maturation at concentrations inhibitory to PI 3-kinase, and that the mechanism for wortmannin-induced maturation involves the activation of a maturation-promoting factor and MAPK [23].
A positive regulatory role for PIP3 in egg activation signaling has been suggested by the finding that bp(V), a potent inhibitor for PTEN phosphoinositide phosphatase, promotes tyrosine phosphorylation of Src and cortical contraction of Xenopus eggs. (Figure 4A-C) The results indicate that the elevation of PIP3 levels in eggs is sufficient for promoting Src-dependent signal transduction for egg activation.
We demonstrated that, in fertilized eggs, Akt was phosphorylated at Thr308. However, we did not detect the phosphorylation of another well-known site, Ser473. This could be because the phosphoSer473-specific antibody employed in this study was not suitable for analysis of the Xenopus Akt protein. However, immunoblotting has demonstrated that the phosphoSer473-specific antibody well recognizes Akt protein in immature Xenopus oocytes and that the phosphoSer473 signals become decreased in progesterone-treated, fully matured oocytes (unpublished results). Therefore, we suggest that fertilization promotes the phosphorylation of Akt selectively at threonine-308. In mammals, phosphorylation of Akt Thr308 and Ser473 is catalyzed by phosphoinositide-dependent protein kinase PDK1 [46] and mammalian target of rapamycin mTOR [47], respectively. So, we assume that the signaling pathway involving PDK1, not mTOR, operates to promote the phosphorylation and activation of Akt in fertilized eggs.
Several cellular functions involve the activation of Akt [46,48]. Of particular interest is the anti-apoptotic function of Akt. Our preliminary experiments have shown that a caspase-like protease is activated in eggs left unfertilized for several hours (unpublished results). The results suggest that fertilization is responsible for suppression of the caspase's activation. Therefore, our future efforts will be directed at examining whether an anti-apoptotic mechanism is really operating in fertilized eggs and, if so, to clarify how and where Akt is involved in this cellular function.
Several studies have focused on the roles of sperm-associated PI 3-kinase in mammalian species. In human sperm, LY294002 blocks the acrosome reaction [49] and enhances motility [50], but does not affect egg-sperm interaction [51]. More recently, Jungnickel et al. [52] demonstrated that in the mouse, the zona pellucida glycoprotein 3-induced acrosome reaction involves the PIP3-dependent activation of Akt. These results suggest that PI 3-kinase is required for Ca2+ signaling in fertilizing sperm. In Xenopus, however, no information has yet been available for the role of PI 3-kinase and/or Akt in the acrosome reaction or other fertilization-related functions of sperm.
Another important question is how PI 3-kinase could be activated after egg-sperm interaction/fusion (without the involvement of tyrosine phosphorylation)? In this connection, the fact that fertilization is accompanied by a transient accumulation of the p85 subunit of PI 3-kinase and Thr308-phosphorylated form of Akt in the egg membrane microdomains may be important. As we reported earlier, egg membrane microdomains are abundant in cholesterol, the ganglioside GM1, and several signaling molecules, and serve as a platform for egg-sperm interaction and subsequent Src-dependent signal transduction [42,53]. More recently, we have identified and characterized a membrane microdomain-associated protein, uroplakin III, which might act as a target of sperm-derived protease and as a substrate of the activated Src at fertilization [16,54,55]. Although the signal transduction pathway connecting uroplakin III and Src is not yet known, the involvement of GTP-binding proteins has been inferred because the application of GTP to the isolated egg membrane microdomains could reconstitute the Src activity in vitro (Iwasaki et al. manuscript in preparation; 53). So, it is tempting to speculate that PI 3-kinase acts as a downstream target of GTP-binding proteins, as demonstrated in the case of γ-subspecies of PI 3-kinase [56], so that GTP- and PIP3-dependent activation of Src is possible.
The specific activation of PI 3-kinase, however, is not necessarily required for egg activation signaling. Rather, the rapid and transient accumulations of PI 3-kinase at membrane microdomains by itself maybe important. Such a local concentration of PI 3-kinase may contribute to the effective propagation of PIP3-dependent and LY294002-sensitive signals for egg activation. In support of this, PIP2 is known to predominantly localize to membrane microdomains of mammalian cells [57,58]. Further study should be directed toward analyzing the mechanism by which membrane microdomains allow the precise localization of these signaling molecules before and after fertilization.
In this study, we show that LY294002 effectively inhibited several early steps of sperm-induced egg activation, including the activation of Src. Although we should keep in mind that the effect of LY294002 could be mediated through other targets than PI 3-kinase, we think that the inhibitory effect of LY294002 is most likely due to the inhibition of PI 3-kinase: because not only several steps of Src-dependent egg activation but also sperm-induced phosphorylation of Akt are inhibited by this inhibitor. PI 3-kinase and Akt become activated and temporarily localize to the egg membrane microdomains where sperm-induced tyrosine kinase machinery operates for successful fertilization. These results highlight for the first time the important role the egg-associated PI 3-kinase plays in signal transduction for Xenopus fertilization.
Animals, antibodies, cultured cells and chemicals
Frogs were purchased from local dealers and maintained as described [11,42]. Rabbit antibody to human Src phosphorylated at Tyr418 was obtained from Oncogene Research Products (CA, USA). Mouse anti-mouse PLCγ antibody was from Upstate Biotechnology (NY, USA). Rabbit antibodies against the recombinant Xenopus UPIII extracellular domain (xUPIII-ED) or a part of the Xenopus Src amino-terminal region were prepared as described previously [54,59]. The mouse anti-phosphotyrosine antibody PY99 was purchased from Santa Cruz (CA, USA). Rabbit antibodies against phosphorylated human MAPK, human cyclin B2, and Xenopus Mos were from BioLabs (MA, USA) or Santa Cruz Biotechnology. Rabbit antibody against the 85-kDa subunit of human PI 3-kinase was obtained from Upstate Cell Signaling Solution (VA, USA). Rabbit antibodies against human Akt, and the phosphorylated Thr308 or phosphorylated Ser473 form of human Akt were obtained from Cell Signaling Technology (MA, USA). Human embryonic kidney 293 cells were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum at 37°C/5% CO2 in a humidified incubator. A Src-specific inhibitor, PP2, its inactive analog, PP3, a PLC-specific inhibitor, U73122, and a PI 3-kinase-specific inhibitor, LY294002, were purchased from Calbiochem. A23187 and bovine spleen cathepsin B were from Sigma (MO, USA). Leupeptin was from Peptide Institute (Osaka, Japan). H2O2 was from Santoku Chemical Industries (Tokyo, Japan). (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (APMSF) was from Calbiochem (CA, USA). A synthetic tyrosine kinase substrate peptide (Cdk1 peptide) was synthesized and purified as described previously [60]. The fluorescent Ca2+ indicator fura-2 was obtained from Calbiochem. Xenopus Src was partially (one column chromatography-processed) or fully (four column chromatography-processed) purified from immature oocytes as described previously [60], and used for kinase assays in vitro (see below). [γ-32P]ATP was obtained from ICN (OH, USA). A potent PTEN (phosphatase and tensin homolog deleted from chromosome 10) inhibitor bp(V) [dipotassium bisperoxo (picolinato) oxovanadate (V)] was purchased from Calbiochem. Phosphatidylinositol (PI) was obtained from Serdary Research Laboratories (Ontario, Canada). PI 4-phosphate and PI 4,5-bisphosphate were prepared from bovine brain as described previously [61]. PI 3,4,5-trisphosphate (fatty acid moieties are palmitate, C16:0) was prepared as described [62]. All the phospholipids were lyophilized from the chloroform solution and suspended in distilled water or 20 mM Tris-HCl (pH 7.5) by sonication. Protein A-Sepharose was obtained from GE Healthcare Biosciences (Uppsala, Sweden). Unless otherwise indicated, other chemicals were purchased from Sigma, Wakenyaku (Kyoto, Japan), Wako (Osaka, Japan), or Nacalai Tesque (Kyoto, Japan).
Eggs, sperm, and embryos
The collection of unfertilized eggs and sperm, removal of the jelly layer from eggs, and jelly water treatment of sperm were carried out as described previously [11,42]. The activation of jelly layer-free eggs was done by insemination with jelly water-treated sperm (106 sperm/ml) or by parthenogenetic activation with the calcium ionophore A23187 (0.5 μM), H2O2 (10 mM), cathepsin B (5 U/ml), or bp(V) (200 μM) for the periods specified in the text. After the activation treatments, egg samples were washed with ice-cold DeBoer's solution containing 110 mM NaCl, 1.3 mM KCl, and 0.44 mM CaCl2, pH 7.2, immediately frozen in liquid nitrogen, and kept at -80°C. Activation was monitored by the occurrence of cortical contraction and first cell cleavage in a group of control eggs.
To determine the requirement of egg PI 3-kinase activity for Xenopus egg activation, the unfertilized eggs were microinjected with LY294002 as described previously [11]. The microinjection of PP2, PP3, U73122, or DMSO alone was also performed. Briefly, stock solutions (5 mM in DMSO) for each compound were diluted with buffer containing 20 mM sodium phosphate, pH 7.4, and 100 mM NaCl. The inhibitor solutions were then loaded into an oil-filled glass capillary (Drummond), with a tip diameter of 10-30 μm, which was connected to a pulse-directed injector system (Nanoject; Drummond). Before being microinjected, dejellied eggs were washed and incubated with injection buffer containing 110 mM NaCl, 1.3 mM KCl, 20 mM MgSO4, 0.1 mM EGTA, and 10 mM chlorobutanol. A quantitative injection of the inhibitor solution (25-50 nl per egg) was conducted under microscopic observation, healed, and the eggs subjected to activation treatment (see above).
Biochemical fractionation
To analyze the presence and/or the phosphorylation status of proteins of interest, egg samples were homogenized or vortex-crushed with 10 μl per egg of ice-cold extraction buffer containing 1% (w/v) Triton X-100, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM β-mercaptoethanol, 1 mM Na3VO4, 10 μg/ml leupeptin, and 20 μM APMSF. The extract was centrifuged at 12,000 g and the supernatant was used as the Triton X-100-solubilized egg extract (note that the extract contained Triton X-100-resistant membranes). In some experiments, egg samples were subjected to the preparation of crude membrane fractions as described [60]. The membrane fractions were solubilized with the extraction buffer containing 0.1% SDS, and subjected to further experiments. Alternatively, eggs were subjected to the preparation of membrane microdomains as described previously [63]. Briefly, eggs were homogenized with a 5-fold volume of ice-cold extraction buffer without Triton X-100. The homogenates devoid of debris were centrifuged at 150,000 g for 1 h. After the centrifugation, the fluffy layer of the pellets, rich in plasma membranes, were collected and adjusted to 1% Triton X-100 (w/v) and 42.5% (w/v) sucrose. The mixtures were then layered with 30% (first) and 5% (second) sucrose in the extraction buffer and centrifuged at 200,000 g for 24 h in an SW55 rotor (Beckman). After the centrifugation, aliquots of 12 fractions were collected from the top to bottom of the tubes. Fractions 3-6 were pooled as low density, detergent-insoluble membrane fractions (membrane microdomains) whereas fractions 9-12 were pooled as detergent-soluble, non-microdomain fractions. Protein concentrations were determined by the dye-binding assay (Bio-Rad, CA, USA).
Immunoprecipitation, immunoblotting, and SDS-PAGE
Protein samples (50-500 μg, 1 mg/ml) were immunoprecipitated with antibodies as specified in the text for 2 h at 4°C. The immune complexes were adsorbed onto 10 μl of protein A-Sepharose beads by gentle mixing for 30 min at 4°C. The beads were washed three times with 500 μl of buffer containing 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM Na3VO4, 10 μg/ml leupeptin, and 20 μM APMSF. The washed beads were treated with Laemmli's SDS sample buffer [64] at 98°C for 5 min. SDS-denatured proteins were separated by SDS-polyacrylamide gel electrophoresis using 10% gels and analyzed by immunoblotting as described previously [42].
In vitro kinase assay
The protein kinase assay was carried out in a standard reaction mixture (25 μl) containing an immunoprecipitated or purified Xenopus Src, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 μM [γ-32P]ATP (3.7 kBq/pmol), 1 mM dithiothreitol, and 1 mM Cdk1 peptide. Anti-pepY antibody, which recognized the autophosphorylation site of Src, and SDS-solubilized egg membrane fractions were used for immunoprecipitation as described previously [43,60]. When the effect of several phospholipids was evaluated, purified Xenopus Src and non-radioactive ATP (at 1 mM) were employed, and Cdk1 peptide was omitted. The reaction was initiated by the addition of [γ-32P]ATP or non-radioactive ATP at 30°C for 10 min and terminated by the addition of SDS-sample buffer [64] followed by boiling for 3 min. Phosphorylation of Cdk1 peptide (radioactive assay) was analyzed by separating the reaction mixtures on 18% SDS-polyacrylamide slab gels, followed by visualization and quantification by a BAS2000 Bioimaging Analyzer (Fuji Film, Tokyo, Japan). Autophosphorylation of Xenopus Src (non-radioactive assay) was analyzed by separating the reaction mixtures by using 10% SDS-polyacrylamide slab gels, followed by immunoblotting with the anti-phosphorylated Tyr418 antibody.
Intracellular Ca2+ measurement
For Ca2+ measurements, dejellied eggs prepared from albino Xenopus females were co-microinjected with 50 nl of 160 μM fura-2 plus 200 μM LY294002 (see "Microinjection"). The injected eggs were then fertilized and fluorescent signals were recorded by ratio-imaging microscopy using a high-frame digital CCD imaging ARGUS/HISCA system (Hamamatsu Photonics, Japan) with a 10× planneofluar objective, NA 0.3 (Zeiss, Germany). Excitation wavelengths of 340 and 380 nm (each irradiation constant was set to about 800 ms) were used while monitoring the emission at 510 nm. All data collections were made at 10-s intervals at 18-21°C.
Authors' contributions
MG acquired the experimental data and prepared a preliminary draft. TI, AAT, and YF participated in the design of the study and contributed critical comments and suggestions while performing experiments and preparing drafts. KS organized the research, obtained data, interpreted results, and revised and finalized the manuscript. All authors read and approved the final manuscript.
This work is supported in part by a Grant-in-Aid for Scientific Research (B), Japan Society for the Promotion of Science (19370094) and a grant from Hyogo Science and Technology Association (19I020) to KS.
Evans JP, Florman HM: The state of the union: the cell biology of fertilization.
Nat Cell Biol 2002, 4(Suppl):s57-63. PubMed Abstract | Publisher Full Text
Horner VL, Wolfner MF: Transitioning from egg to embryo: triggers and mechanisms of egg activation.
Dev Dyn 2008, 237:527-544. PubMed Abstract | Publisher Full Text
Runft LL, Jaffe LA, Mehlmann LM: Egg activation at fertilization: where it all begins.
Dev Biol 2002, 245:237-254. PubMed Abstract | Publisher Full Text
Ducibella T, Fissore R: The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development.
Dev Biol 2008, 315:257-279. PubMed Abstract | Publisher Full Text
Miyazaki S, Shirakawa H, Nakada K, Honda Y: Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs.
Dev Biol 1993, 158:62-78. PubMed Abstract | Publisher Full Text
Whitaker M: Calcium at fertilization and in early development.
Physiol Rev 2006, 86:25-88. PubMed Abstract | Publisher Full Text
Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K, Lai FA: PLCζ: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development.
Development 2002, 129:3533-3544. PubMed Abstract | Publisher Full Text
Harada Y, Matsumoto T, Hirahara S, Nakashima A, Ueno S, Oda S, Miyazaki S, Iwao Y: Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster.
Dev Biol 2007, 306:797-808. PubMed Abstract | Publisher Full Text
Abassi YA, Carroll DJ, Giusti AF, Belton RJ Jr, Foltz KR: Evidence that Src-type tyrosine kinase activity is necessary for initiation of calcium release at fertilization in sea urchin eggs.
Dev Biol 2000, 218:206-219. PubMed Abstract | Publisher Full Text
Giusti AF, Carroll DJ, Abassi YA, Terasaki M, Foltz KR, Jaffe LA: Requirement of a Src family kinase for initiating calcium release at fertilization in starfish eggs.
J Biol Chem 1999, 274:29318-29322. PubMed Abstract | Publisher Full Text
Sato K, Tokmakov AA, Iwasaki T, Fukami Y: Tyrosine kinase-dependent activation of phospholipase Cγ is required for calcium transient in Xenopus egg fertilization.
Dev Biol 2000, 224:453-469. PubMed Abstract | Publisher Full Text
Shearer J, De Nadai C, Emily-Fenouil F, Gache C, Whitaker M, Ciapa B: Role of phospholipase Cγ at fertilization and during mitosis in sea urchin eggs and embryos.
Development 1999, 126:2273-84. PubMed Abstract | Publisher Full Text
Sharma D, Kinsey WH: Fertilization triggers localized activation of Src-family protein kinases in the zebrafish egg.
Dev Biol 2006, 295:604-614. PubMed Abstract | Publisher Full Text
Wu W, Kinsey WH: Fertilization triggers activation of Fyn kinase in the zebrafish egg.
Int J Dev Biol 2000, 44:837-841. PubMed Abstract | Publisher Full Text
Iwao Y, Fujimura T: Activation of Xenopus eggs by RGD-containing peptides accompanied by intracellular Ca2+ release.
Dev Biol 1996, 177:558-567. PubMed Abstract | Publisher Full Text
Mahbub Hasan AK, Sato K, Sakakibara K, Ou Z, Iwasaki T, Ueda Y, Fukami Y: Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization.
Dev Biol 2005, 286:483-492. PubMed Abstract | Publisher Full Text
Mizote A, Okamoto S, Iwao Y: Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization.
Dev Biol 1999, 208:79-92. PubMed Abstract | Publisher Full Text
Shilling FM, Magie CR, Nuccitelli R: Voltage-dependent activation of frog eggs by a sperm surface disintegrin peptide.
Dev Biol 1998, 202:113-124. PubMed Abstract | Publisher Full Text
Andersen CB, Sakaue H, Nedachi T, Kovacina KS, Clayberger C, Conti M, Roth RA: Protein kinase B/Akt is essential for the insulin- but not progesterone-stimulated resumption of meiosis in Xenopus oocytes.
Biochem J 2003, 369:227-238. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Liu XJ, Sorisky A, Zhu L, Pawson T: Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.
Mol Cell Biol 1995, 15:3563-3570. PubMed Abstract | PubMed Central Full Text
Muslin AJ, Klippel A, Williams LT: Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation.
Mol Cell Biol 1993, 13:6661-6666. PubMed Abstract | PubMed Central Full Text
Hehl S, Stoyanov B, Oehrl W, Schönherr R, Wetzker R, Heinemann SH: Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.
Biochem J 2001, 360:691-698. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Carnero A, Lacal JC: Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, induces oocyte maturation through a MPF-MAPK-dependent pathway.
FEBS Lett 1998, 422:155-159. PubMed Abstract | Publisher Full Text
Lambert CC: Signaling pathways in ascidian oocyte maturation: effects of various inhibitors and activators on germinal vesicle breakdown.
Dev Growth Differ 2005, 47:265-272. PubMed Abstract | Publisher Full Text
Hoshino Y, Yokoo M, Yoshida N, Sasada H, Matsumoto H, Sato E: Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes.
Mol Reprod Dev 2004, 69:77-86. PubMed Abstract | Publisher Full Text
Kalous J, Solc P, Baran V, Kubelka M, Schultz RM, Motlik J: PKB/AKT is involved in resumption of meiosis in mouse oocytes.
Biol Cell 2006, 98:111-123. PubMed Abstract | Publisher Full Text
Hirohashi N, Harada K, Chiba K: Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish.
Dev Biol 2008, 318:194-202. PubMed Abstract | Publisher Full Text
Reddy P, Liu L, Adhikari D, Jagarlamudi K, Rajareddy S, Shen Y, Du C, Tang W, Hämäläinen T, Peng SL, Lan ZJ, Cooney AJ, Huhtaniemi I, Liu K: Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool.
Science 2008, 319:611-613. PubMed Abstract | Publisher Full Text
Browaeys-Poly E, Cailliau K, Vilain JP: Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cγ.
Eur J Biochem 2000, 267:6256-6263. PubMed Abstract | Publisher Full Text
Browaeys-Poly E, Cailliau K, Vilain JP: Transduction cascades initiated by fibroblast growth factor 1 on Xenopus oocytes expressing MDA-MB-231 mRNAs. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cγ.
Cell Signal 2001, 13:363-368. PubMed Abstract | Publisher Full Text
Gould GW, Jess TJ, Andrews GC, Herbst JJ, Plevin RJ, Gibbs EM: Evidence for a role of phosphatidylinositol 3-kinase in the regulation of glucose transport in Xenopus oocytes.
J Biol Chem 1994, 269:26622-26625. PubMed Abstract | Publisher Full Text
Salaün P, Le Breton M, Morales J, Bellé R, Boulben S, Mulner-Lorillon O, Cormier P: Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos.
Exp Cell Res 2004, 296:347-357. PubMed Abstract | Publisher Full Text
De Nadai C, Huitorel P, Chiri S, Ciapa B: Effect of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, on the first mitotic divisions of the fertilized sea urchin egg.
J Cell Sci 1998, 111:2507-2518. PubMed Abstract | Publisher Full Text
Li Y, Chandrakanthan V, Day ML, O'Neill C: Direct evidence for the action of phosphatidylinositol (3,4,5)-trisphosphate-mediated signal transduction in the 2-cell mouse embryo.
Biol Reprod 2007, 77:813-821. PubMed Abstract | Publisher Full Text
Carballada R, Yasuo H, Lemaire P: Phosphatidylinositol-3 kinase acts in parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm induction.
Development 2001, 128:35-44. PubMed Abstract | Publisher Full Text
Nie S, Chang C: PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation.
Mech Dev 2007, 124:657-667. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ueno S, Kono R, Iwao Y: PTEN is required for the normal progression of gastrulation by repressing cell proliferation after MBT in Xenopus embryos.
Dev Biol 2006, 297:274-283. PubMed Abstract | Publisher Full Text
Peng Y, Jiang BH, Yang PH, Cao Z, Shi X, Lin MC, He ML, Kung HF: Phosphatidylinositol 3-kinase signaling is involved in neurogenesis during Xenopus embryonic development.
J Biol Chem 2004, 279:28509-28514. PubMed Abstract | Publisher Full Text
Mehlmann LM, Chattopadhyay A, Carpenter G, Jaffe LA: Evidence that phospholipase C from the sperm is not responsible for initiating Ca2+ release at fertilization in mouse eggs.
Dev Biol 2001, 236:492-501. PubMed Abstract | Publisher Full Text
Carpenter CL, Cantley LC: Phosphoinositide kinases.
Curr Opin Cell Biol 1996, 8:153-158. PubMed Abstract | Publisher Full Text
Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC, Woscholski R, Parker PJ, Waterfield MD: Synthesis and function of 3-phosphorylated inositol lipids.
Annu Rev Biochem 2001, 70:535-602. PubMed Abstract | Publisher Full Text
Sato K, Iwasaki T, Ogawa K, Konishi M, Tokmakov AA, Fukami Y: Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization.
Development 2002, 129:885-896. PubMed Abstract | Publisher Full Text
Sato K, Iwao Y, Fujimura T, Tamaki I, Ogawa K, Iwasaki T, Tokmakov AA, Hatano O, Fukami Y: Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.
Dev Biol 1999, 209:308-320. PubMed Abstract | Publisher Full Text
Sato K, Fukami Y, Stith BJ: Signal transduction pathways leading to Ca2+ release in a vertebrate model system: lessons from Xenopus eggs.
Semin Cell Dev Biol 2006, 17:285-292. PubMed Abstract | Publisher Full Text
Rameh LE, Chen CS, Cantley LC: Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins.
Cell 1995, 83:821-830. PubMed Abstract | Publisher Full Text
Brazil DP, Yang ZZ, Hemmings BA: Advances in protein kinase B signalling: AKTion on multiple fronts.
Trends Biochem Sci 2004, 29:233-242. PubMed Abstract | Publisher Full Text
Hresko RC, Mueckler M: mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes.
J Biol Chem 2005, 280:40406-40416. PubMed Abstract | Publisher Full Text
Parcellier A, Tintignac LA, Zhuravleva E, Hemmings BA: PKB and the mitochondria: AKTing on apoptosis.
Cell Signal 2008, 20:21-30. PubMed Abstract | Publisher Full Text
Fisher HM, Brewis IA, Barratt CL, Cooke ID, Moore HD: Phosphoinositide3-kinase is involved in the induction of the human sperm acrosome reaction downstream of tyrosine phosphorylation.
Mol Hum Reprod 1998, 4:849-855. PubMed Abstract | Publisher Full Text
Luconi M, Marra F, Gandini L, Filimberti E, Lenzi A, Forti G, Baldi E: Phosphatidylinositol 3-kinase inhibition enhances human sperm motility.
Hum Reprod 2001, 16:1931-1937. PubMed Abstract | Publisher Full Text
Barbonetti A, Zugaro A, Sciarretta F, Santucci R, Necozione S, Ruvolo G, Francavilla S, Francavilla F: The inhibition of the human sperm phosphatidylinositol 3-kinase by LY294002 does not interfere with sperm/oocyte interaction.
Int J Androl 2006, 29:468-474. PubMed Abstract | Publisher Full Text
Jungnickel MK, Sutton KA, Wang Y, Florman HM: Phosphoinositide-dependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction.
Dev Biol 2007, 304:116-126. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sato K, Tokmakov AA, He CL, Kurokawa M, Iwasaki T, Shirouzu M, Fissore RA, Yokoyama S, Fukami Y: Reconstitution of Src-dependent phospholipase Cγ phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs.
J Biol Chem 2003, 278:38413-38420. PubMed Abstract | Publisher Full Text
Sakakibara K, Sato K, Yoshino K, Oshiro N, Hirahara S, Mahbub Hasan AK, Iwasaki T, Ueda Y, Iwao Y, Yonezawa K, Fukami Y: Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization.
J Biol Chem 2005, 280:15029-15037. PubMed Abstract | Publisher Full Text
Mahbub Hasan AK, Ou Z, Sakakibara K, Hirahara S, Iwasaki T, Sato K, Fukami Y: Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction.
Genes Cells 2007, 12:251-267. PubMed Abstract | Publisher Full Text
Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nürnberg B: Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.
Science 1995, 269:690-693. PubMed Abstract | Publisher Full Text
Liu Y, Casey L, Pike LJ: Compartmentalization of phosphatidylinositol 4,5-bisphosphate in low-density membrane domains in the absence of caveolin.
Biochem Biophys Res Commun 1998, 245:684-690. PubMed Abstract | Publisher Full Text
Pike LJ, Casey L: Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains.
J Biol Chem 1996, 271:26453-26456. PubMed Abstract | Publisher Full Text
Iwasaki T, Sato K, Yoshino K, Itakura S, Kosuge K, Tokmakov AA, Owada K, Yonezawa K, Fukami Y: Phylogeny of vertebrate Src tyrosine kinases revealed by the epitope region of mAb327.
Sato K, Aoto M, Mori K, Akasofu S, Tokmakov AA, Sahara S, Fukami Y: Purification and characterization of a Src-related p57 protein-tyrosine kinase from Xenopus oocytes. Isolation of an inactive form of the enzyme and its activation and translocation upon fertilization.
J Biol Chem 1996, 271:13250-13257. PubMed Abstract | Publisher Full Text
Sato K, Yamamoto H, Otsuki T, Aoto M, Tokmakov AA, Hayashi F, Fukami Y: Phosphatidylinositol 4,5-bisphosphate stimulates phosphorylation of the adaptor protein Shc by c-Src.
FEBS Lett 1997, 410:136-140. PubMed Abstract | Publisher Full Text
Sato K, Otsuki T, Kimoto M, Kakumoto M, Tokmakov AA, Watanabe Y, Fukami Y: c-Src and phosphatidylinositol 3-kinase are involved in NGF-dependent tyrosine phosphorylation of Shc in PC12 cells.
Biochem Biophys Res Commun 1998, 250:223-228. PubMed Abstract | Publisher Full Text
Sato K, Yoshino K, Tokmakov AA, Iwasaki T, Yonezawa K, Fukami Y: Studying fertilization in cell-free extracts: focusing on membrane/lipid raft functions and proteomics.
Methods Mol Biol 2006, 322:395-411. PubMed Abstract | Publisher Full Text
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature 1970, 227:680-685. PubMed Abstract | Publisher Full Text


text:
Campbell's Kitchen
Original recipe yields 8 servings
Ready In
This was a nice, warm, filling meal but nothing out of the ordinary. The topping was really yummy though.
It was a good way to use up left-over chicken. I agree that there were way too many dished involved- I had a whole sinkful by the time I was finished. I did substitute some whole wheat pasta and...
This was a hit! I followed the directions exactly, except that I used two cups of stuffing mix for the topping and substitued a broccoli/caulifower mix instead of the carrots and peas. Very go...

subset: commoncrawl


text:
Altera Updates First Quarter Revenue Guidance
PR Newswire
SAN JOSE, Calif., March 8, 2012
SAN JOSE, Calif., March 8, 2012 /PRNewswire/ -- Altera Corporation (NASDAQ: ALTR) today announced updated first quarter 2012 revenue guidance.
Initial guidance for the first quarter was for a 5 to 9 percent sequential revenue decline, largely the result of program timing in the military vertical market and continued softness in sales to wireless customers. As the quarter has progressed, the company has experienced somewhat more pronounced and broader than anticipated inventory adjustment related weakness and now believes that first quarter revenue will be 7 to 9 percent lower than fourth quarter levels.
The company continues to expect that revenue in the second quarter of 2012 will be above first quarter levels. Quarter-to-date book to bill remains above 1.0.
First quarter results will be released after the market close on April 19, 2012.
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Statements in this press release that are not historical are "forward-looking statements" as the term is defined in the Private Securities Litigation Reform Act of 1995. Forward-looking statements are generally written in the future tense and/or preceded by words such as "will," "expects," "anticipates," or other words that imply or predict a future state. Forward-looking statements include, but are not limited to, the company's first and second quarter revenue outlook. Investors are cautioned that all forward-looking statements in this release involve risks and uncertainty that can cause actual results to differ from those currently anticipated, due to a number of factors, including without limitation, current global economic conditions, customer business environment, customer inventory levels, vertical market mix, market acceptance of the company's products, product introduction schedules, the rate of growth of the company's new products including Cyclone® IV, Arria® II, Arria V, Stratix® IV, and Stratix V FPGAs, MAX® V CPLDs and HardCopy® IV device families, changes in the mix of our business between prototyping and production-based demand, as well as changes in economic conditions and other risk factors discussed in documents filed by the company with the Securities and Exchange Commission (SEC) from time to time. Copies of Altera's SEC filings are posted on the company's website and are available from the company without charge. Forward-looking statements are made as of the date of this release, and, except as required by law, the company does not undertake an obligation to update its forward-looking statements to reflect future events or circumstances.About Altera
Altera programmable solutions enable system and semiconductor companies to rapidly and cost-effectively innovate, differentiate and win in their markets. Find out more about Altera's FPGA, CPLD and ASIC devices at www.altera.com. Follow Altera via Facebook, RSS and Twitter.
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The Upstate Club will host our 2015 Firecracker Fling on July 4-5! This is two SEPARATE one-day, PDGA C-Tier tournaments! Saturday will be held at JB Red Owens in Easley, and Sunday will be held at Tyger River Park in Duncan. See the flyer for complete details. You must register for each day separately through Disc Golf United - Saturday and Sunday.
SC Juniors Championship
The Upstate Club's 2015 SC Junior State Championships will be on August 1 at Grand Central Station. This competition is only for players 19 years old or younger. Separate divisions will be offered based on age and gender. See the flyer for complete details. Please register online through Disc Golf United.
2015 Club Membership
Join the Upstate Disc Golf Club as we work to continually grow and improve disc golf in the upstate. Membership is updated on a calendar-year basis.
- Basic Membership - $10
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- Club stamped mini (first-time members only)
- 2015 club t-shirt
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- $5 discount on the entry fees to The Melee and Glow Doubles
- Ability to spend merchandise vouchers on non-club merchandise
- Free entry to the end-of-year club party


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Study on Forest Dependent Households under a Household Model Framework
This study suggests that JFM households receive higher economic benefit after JFM: the physical increase of forest related works has a positive impact on the prices of the same influencing higher hours (time) of work which help them increase higher annual per capita net real income. The poorer the households are according to their economic status, greater is the dependence on forest and so greater is the extent of involvement in low return forest activities (NTFPs) and forest wage work. It might indicate that JFM plays a positive role for economic security of the forest fringe households.
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- Baland, Jean-Marie & Platteau, Jean-Philippe, 2000. "Halting Degradation of Natural Resources: Is There a Role for Rural Communities?," OUP Catalogue, Oxford University Press, number 9780198290612, March.
- Kelvin J. Lancaster, 1966. "A New Approach to Consumer Theory," Journal of Political Economy, University of Chicago Press, vol. 74, pages 132.
- Sarker, Debnarayan & Das, Nimai, 2004. "Social Capital and Joint Forest Management Programme: A Comparative Study on Female-headed and Joint Forest Protection Committees in West Bengal," MPRA Paper 14780, University Library of Munich, Germany, revised 2005.
- Debnarayan Sarker & Nimai Das, 2008.
"A Study of Economic Outcome of Joint Forest Management Programme in West Bengal: The Strategic Decisions between Government and Forest Fringe Community,"
Indian Economic Review,
Department of Economics, Delhi School of Economics, vol. 43(1), pages 17-45, July.
- Sarker, Debnarayan & Das, Nimai, 2006. "A Study of Economic Outcome of Joint Forest Management Programme in West Bengal: The Strategic Decisions between Government and Forest Fringe Community," MPRA Paper 14875, University Library of Munich, Germany, revised 2007.
- Sarker, Debnarayan & Das, Nimai, 2006. "Efficiency of Market Behaviour of NTFPs for Households under JFMP: A Case Study in West Bengal," MPRA Paper 14779, University Library of Munich, Germany, revised 2007.
- Pattanayak, Subhrendu K. & Sills, Erin O. & Kramer, Randall A., 2004. "Seeing the forest for the fuel," Environment and Development Economics, Cambridge University Press, vol. 9(02), pages 155-179, May.
- Fisher, Monica, 2004. "Household welfare and forest dependence in Southern Malawi," Environment and Development Economics, Cambridge University Press, vol. 9(02), pages 135-154, May.
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What to expect:
At the Parks at Chehaw, an 800-acre recreation facility, you can camp amid piney woods near a wild-animal park/zoo where elephants, zebras, and cheetahs roam in naturalistic habitats. The animals won't come sniffing around your tent, though, since they are separated by fences from humans. But you and your child can visit them whenever you want during the day via a series of elevated boardwalks and paths that lead through the pines and cypress swamps, allowing for close-up views of the animals, which include both exotic and native species.
Preschoolers may enjoy the park's petting zoo just as much as spotting the big animals. There's a building that houses reptiles and small animals, too. A nature trail winds gently alongside Muckalee Creek, where you may spot white-tailed deer, squirrels, beavers, tortoises, foxes, birds — even alligators (they're not close enough to be dangerous).
The Parks at Chehaw offer a campground, which was voted one of America's Choicest Campgrounds by Nick, Jr. Magazine. The campground is open every day year-round and is suitable for tents, trailers, or RVs. The campground has reasonable rates, and visiting the wild-animal park won't put a big dent in your pocketbook. For families with young children who are fascinated by animals, it all adds up to an unusual, inexpensive outdoor vacation.
And the animals aren't the only draw. Young kids can head for two playgrounds, both within easy walking distance of the campground. The 15,000-square-foot Play Park has an adventure theme with a jungle fortress, a rocket, a ship, a tree house, and an elephant-trunk slide tame enough for even the tiniest tots. Young children may also thrill to riding a miniature train, the Wiregrass Express (a replica of an 1863 model), which winds through the campground and other areas of the parks on a 3.2-mile, 20-minute journey. The scenery is mostly woods, but the train itself offers plenty of fun.
Forty-six campsites with water and electrical hookups, laundry facilities, rest rooms, bathhouse, picnic pavilions. On nearby Highway 91, you can purchase gas, picnic supplies, and snacks. The wild-animal park has a concession stand with snacks and a gift shop.
- Each campsite is for one immediate family, plus up to two cars and a tent or RV. Maximum stay is 14 consecutive days.
- The Parks at Chehaw sponsor a variety of special events throughout the year, including Native American festivals and Halloween celebrations. Visit Parks at Chehaw website or call (229) 430-5275 for a schedule.
The campground is open daily, year-round. Southern Georgia tends to stay fairly warm throughout the year, but it does sometimes get cold in winter. The animal park is closed on Thanksgiving, Christmas, and New Year's Day.
Getting there:
The campground is about three hours (184 miles) south of Atlanta. Take I-75 south to Exit 32 (Highway 300). Go south on Highway 300, then turn west on Highway 32. Follow Highway 32 and turn left onto Highway 91 (Philema Road); go nine miles to the park, which is on the right.
For rates, reservations, and more information visit Parks at Chehaw website or call (229) 430-5275 or. The campground does not require reservations, but you might want to call ahead, especially for holiday weekends and other peak vacation times.


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CONDOMINIUM UNIT: 3 bed, 2 full bath, 1 half bath. Unique opportunity to own this beautiful Ocean View, Townhome style Condo in Oceanfront complex locMore info
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Pronunciation: derailleur
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Includes 1.59 Index Single Vision Lenses
True Religion Geno Dark Brown Cocoa eyeglasses are contemporary yet refined. This demure style has a distinguished rectangular shape paired with a satin brown metal composition for a classic combination. The glossy, dark brown acetate temples highlight the golden True Religion signature embedded by the hinges and polished logos at the tips.About the Brand
Inspired by the bohemian-chic and hippie style, True Religion Eyewear brings in the trendiness and everyday living style. Their designs are meant to be comfortable for everyone that wants to walk with style every day.


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42 years of experience
Video profile
Accepting new patients
42 years of experience
Video profile
Accepting new patients
1600 SE Court Pl
Ste L01
Pendleton, OR 97801 Phone number & directions
Ste L01
Pendleton, OR 97801 Phone number & directions
License & Identifications
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Donald Guenther:
View the full profile to get information on:
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The overall average patient rating of Dr. Donald Guenther is Average.
Specialties & Research
Dr. Donald Guenther, MD is board certified in Pediatrics.
Education & Awards
Dr. Guenther graduated from University of California at Irvine.
Dr. Guenther has 1 hospital affiliations:
- St Anthony Hospital
Home > Find a Pediatrician > OR > Pendleton Pediatricians > Dr. Donald Guenther, MD
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- Dr. Donald Clifford Guenther MD is a male Pediatrician, has 42 years of experience and practices in Pediatrics.
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- Dr. Donald Guenther has 1 office location.
Dr. Donald Guenther practices Pediatrics near Pendleton, OR.
See Pendleton Pediatricians.


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June 16th, 2008 Laura Posted in NEW RELEASES 24 Comments »
June 14th, 2008 Laura Posted in NEW RELEASES 25 Comments »
June 13th, 2008 Laura Posted in NEW RELEASES 18 Comments »
BLACK BULLET (Manga), Vol. 1
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story by Shidu Jin, illustration by Mahiro Satou
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story Kazuma Kamachi, art by Chuya Kogino, character design by Kiyotaka Haimura
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Kanye West Admits: 'I Fell in Love With Kim' Kardashian
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Lovesick Kanye!
On Wednesday, Kanye West unexpectedly released a brand-new song online, "Theraflu," and in between the 34-year-old rapper's typically over-the-top boasts and X-rated tangents, he makes a surprising confession about his pal Kim Kardashian. (Listen to the song here -- warning, explicit content.)
Looking back on his romance with ex Amber Rose, who is now engaged to Wiz Khalifa, West raps, "Only n---- I got respect for is Wiz, And I admit I fell in love with Kim around the same time she fell in love with him."
PHOTOS: Kim's less-than-awesome 2011
West then goes on to make a catty reference to Kardashian's future ex-husband, NBA star Kris Humphries, who just happens to play for the New Jersey Nets, which are owned by his good friend Jay-Z. "Well that's cool, baby girl, do your thing, lucky I ain't have Jay drop him from the team," he raps, implying he could get Humphries, 27, fired from the team.
PHOTOS: Kim and Kris' hot-and-heavy honeymoon
Although they've never commented on a romance, West and Kardashian, 31, have been friends for years, and the "Otis" rapper filmed a cameo on "Kourtney & Kim Take New York" in summer 2010. They were spotted hanging out together during Paris Fashion Week earlier this year.
Hot Gossip: Kanye 'Obsessed' With Kim Kardashian? Plus, Hamm-Gate and a Topless Pic
Rumor Control: Kim K. Addresses Kanye Romance, Adoption Report
Hot Gossip: Kim K. Branded a 'Home Wrecker' by Kanye's Blabby Ex
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Inside Gustavus (October 3, 2013)
Volume 46, Number 5
Faculty/Staff Activities
Baker Lawley (English), along with fellow authors and writing professors Tommy Zurhellen (Marist College) and Jody Gehrman (Mendocino College), has co-founded a weekly podcast focusing on the teaching and practice of fiction writing. The podcast is titled Fiction School, and can be found on iTunes or on the web at fictionschool.com.
Doug Huff (Philosophy) was invited to attend a staged-reading of his play, A FAR SHORE (1st Runner-Up of the 2013 Woodward Interntional Playwriting Competition) at the University of New Hampshire (standing ovation) on Sept. 23.
Lynnea Myers (Nursing) presented a poster titled "Nursing Student Counselors' Perceptions of their Role in a Summer Institute of Nursing" at the Sigma Theta Tau Mu Lambda Chapter Annual Research Forum on Wednesday, Sept. 25. The poster highlighted research Lynnea conducted exploring the possible motivations for students to participate in teaching roles such as that of a counselor through the Institute and the relationship to a potential future role in academic or clinical nursing education. Additionally, Lynnea served as a faculty sponsor at the forum for two Gustavus Nursing student presenters, Madeline Randall and Joshua Heinzen, who presented podium sessions on their independent student research projects. Madeline's presentation was titled "An Exploration of the Perceptions of High School Students Related to the Profession of Nursing" and Joshua's presentation was titled "Compassion Fatigue in Rural Midwestern Nurses". Joshua co-presented with a student researcher at St. Olaf College and her faculty advisor, Nicole Beckmann '05.
Andy Biedermann (Print and Mail Services) received the 2013 Distinctive Service Award at the Association of College and University Printers annual conference. The award recognizes individuals and Colleges that innovative goals for growth while meeting continued production demands.
In The Media
- The Mankato Free Press printed a story on Sunday, Sept. 29 about the Gustavus football team's 45-19 victory over St. Olaf. Student-athletes Jeffrey DuBose and Zach Dilger were both quoted in the story.
- The website boingboing.net posted a story on Sept. 30 about The Nobel Conference with a link to the live stream.
- Nobel Conference presenter and Rydell Professor S. James Gates appeared on Minnesota Public Radio's morning show The Daily Circuit on Monday, Sept. 30.
- Senior Cam Michelsen was quoted in an Oct. 1 USA Today story with the headline "Are men's centers essential for college campuses?"
- KEYC ran a story on its Tuesday, Oct. 1 newscasts about The Nobel Conference. Director of Media Relations Matt Thomas and several attendees were interviewed for the story.
- The Mankato Free Press printed front page stories on Wednesday, Oct. 2 about the first two lectures, given by Frank Wilczek and Tara Shears, on day one of the Nobel Conference.
- The Mankato Free Press printed stories on Thursday, Oct. 3 about the Nobel Conference lectures given by George Smoot and Larry Krauss.
- Gustavus alumna Katie DeSantis was featured in an Oct. 3 story in The Washington Informer, a weekly newspaper in Washington, D.C., that serves the African American population.
- Ashley DuBose, who attended Gustavus for her freshman and sophomore years, and was a member of the Class of 2011, auditioned on the NBC show The Voice this week. DuBose was selected to continue competing on the show and is on Adam Levine's team.
New Faces
The following people have recently joined the Gustavus community:
* New Administrators
- Christopher Duhaime, Assistant Manager for the Annual Fund
From the President's Desk
I would like to thank the entire Gustavus community for yet another
successful Nobel Conference! I have learned and come to appreciate
over the past six years that it truly is a campus-wide effort to
pull off an event like The Nobel Conference. This event is part of
what makes Gustavus such a special place and we should all be very
proud of the fact that our hard work reflects well not only on the
Conference, but on the College as a whole.
I especially want to thank Dr. Chuck Niederriter and Dr. Steve Mellema for their leadership as Director and Chair of this year's conference. Dr. Niederriter has done an exceptional job in his position the past five years and has taken great care of the Conference as he now passes the torch on to our next Director, Dr. Scott Bur. I also want to thank all those who coordinated the logistics for the Conference and our faculty and students who hosted the presenters and helped coordinate the visits of our distinguished guests. Gusties Will Shine!
President's Cabinet Summary
The President's Cabinet met on Tuesday, Sept. 24 and discussed the following items:
- President Ohle breifly reviewed the agendas for the Sept. 25 Crown Council meeting and the Sept. 26-27 Board of Trustees meeting.
- A number of academic affairs issues were discussed, including the plans for the shared governance discussions among the Board of Trustees, faculty, and administrators; restricted fund proposal from the department chairs; updating and aligning Commission Gustavus 150 and the Academic Strategic Plan; faculty's willingness to collaborate with Institutional Advancement and Marketing and Communication in "telling the Gustavus story;" faculty grant-writing activity; and return on investment (ROI)/return on a Gustavus education (ROE) conversations.
News & Announcements
Employee Award Nominations...Nominations for the Augusta Carlson Schultz (outstanding support staff employee) and the Eric Norelius (outstanding administrative employee) awards should be turned in to the Office of Human Resources by Oct. 18.
Campus Happenings
Employee/Student Christian Fellowship Luncheon...Tuesday, Oct. 8 from 12-1 p.m. in Linner Lounge. The Gustavus Employee Fellowship group is hosting a lunch for leaders/reps from various Christian student organizations on campus. We are faculty, staff and administrators who gather monthly for fellowship and prayer. This lunch, provided by the Chaplains' Office, is an opportunity to connect with student groups.
LineUs Improv Comedy Show...The LineUs Improv Comedy Troupe is turning 10 years old and to celebrate they're putting on a special performance featuring current and former cast members going all the way back to the troupe's original players. The show is free and will start at 7 p.m. on Saturday, Oct. 5 in Alumni Hall.
African Studies Lecture...University of Minnesota professor Dr. Abdi Samatar will present a lecture titled "The Politics of Piracy in Somalia" at 7 p.m. Monday, Oct. 7 in Wallenberg Auditorium. Dr. Samatar's research focuses on the relationship between democracy and development in the Third World in general and Africa in particular. The event is free and open to the public.
October 8 Lecture...Dr. Toni Calasanti, Professor of Sociology at Virginia Tech University, will speak at Gustavus on Tuesday, Oct. 8 at 4:30 p.m. in Room 101 of Beck Hall. Professor Calasanti's work challenges feminist thinking by highlighting age as a core site of social inequality. In her campus talk, she will show how taking age relationsinto account transforms what we know about gender. In particular, she will share some surprising findings from her research on spousal caregivers to make clear why age matters when we look at gender and care. Lecture is co-sponsored by the departments of economics and management and gender, women, and sexuality studies. The event is free and open to the public.
Gustie Breakfast...The St. Peter Gustie Breakfast will take place at 7:30 a.m. Wednesday, Oct. 9 in a Campus Center banquet room. This month's event features Pearl Leonard-Rock and Glen Lloyd from the Diversity Center. Cost is $8 for an individual or $15 per couple. RSVP to 507-933-6664 or email@example.com.
LALACS Lecture...Francisco Cerezo, a founding member of Comite Cerezo, an organization committed to promoting and defending the human rights of victims of political repression in Mexico, will speak at Gustavus on Wednesday, Oct. 9. His lecture is titled "The U.S., Drugs, and Guns in Mexico: Lessons from the Mexican Human Rights Organization Comite Cerezo" and will take place at 7 p.m. in Room 127 of Confer Hall. His lecture is sponsored by LALACS, Peace Studies, Sociology/Anthropology, and Political Science.
Bards in the Arb Visiting Writers Series...Join the English and Scandinavian Studies departments for an evening filled with original poetry and folk music from Bart and Ross Sutter. Event is from 7 to 9 p.m. on Tuesday, Oct. 8 in the Melva Lind Interpretive Center.
Daily Sabbath...All are invited to the worship services at 10 a.m. weekdays. The upcoming schedule is as follows.
- Friday, Oct. 4 (Play): Commemoration of St. Francis with blessing of the animals.
- Monday, Oct. 7 (Pray): Digital prayer wall, prayers of the heart, prayers for the world. Post your prayers online. Music and silent prayer in Christ Chapel. Silent prayer in the Interfaith Space in the Campus Center.
- Tuesday, Oct. 8 (Discuss): Associate Provost and Dean Darrin Good will provoke the question "How can we put more "co" in the Gustavus "COmmunity?"
- Wednesday, Oct. 9 (Celebrate): Morning praise with the Choir of Christ Chapel
- Thursday, Oct. 10 (Re:vive): Worship led by Re:vive, Chaplain Grady St. Dennis preaching
- Friday, Oct. 11 (Play): To receive mobile updates from the Chaplains, text @GACCHAPLAINS to 23559.
Shared Governance Discussion...All members of the college community are invited to attend this open meeting, hosted in conjunction with the Board of Trustees, in order to contirbute their viewpoints as we work toward a shared understanding of shared governance at Gustavus. This discussion will take place between 2:30 and 4 p.m. Friday, Oct. 11 in the Board Room of the Jackson Campus Center.
Family Weekend...Family Weekend will take place Oct. 11-13 with a number of activities planned. Families are invited to attend classes throughout the day on Friday, Oct. 11. Pancake breakfasts will be hosted on Saturday, Oct. 12 in Norelius, Pittman and Sohre Halls. Workshops and panel discussions will take place on Oct. 12 between 9:30 and 10:30 a.m. in the Jackson Campus Center banquet rooms. Fall Fest in the Arb will take place between 11 a.m. and 5 p.m. in the Linnaeus Arboretum on Oct. 12. A Family Weekend Worship Service will take place at 10 a.m. on Sunday, Oct. 13 in Christ Chapel. The Family Weekend Music Showcase will take place between 2 and 4 p.m. on Oct. 13 in Christ Chapel. Visit the College Calendar for a full list of Family Weekend events and activities.
TIAA-CREF Individual Counseling Sessions…A TIAA-CREF representative will be on campus on October 29, 30, and 31, 2013 for individual counseling sessions. Go online to schedule an appointment, or contact (866) 843-5640, Monday through Friday, from 8 a.m. to 7 p.m.
Congregational Outreach
Retreats...Church Relations in the Center for Servant Leadership will host weekend confirmation retreats for First Lutheran Church of Le Sueur and Woodlake Lutheran Church of Richfield on Saturday, Oct. 5. The confirmation and retreat programs are a resource offered for youth of ELCA congregations who are members of the Gustavus Association of Congregations.
Exploring Religious Questions Series..."World Christianity: The Growth of Christianity in Contemporary Asia, Latin America, and Africa". This mini-course begins on Monday, Oct. 7 from 7-9 p.m. in Olin 103. The beginning session will be led by Marcia Bunge and will focus on introducing the subject of World Christianity by providing an overview of areas in which Christianity is rapidly growing today and by examining new theories about the reasons for and the significance of this growth. Exploring Religious Questions is sponsored by Church Relations in the Center for Servant Leadership.
Safety Snips
Campus Fire Safety Month...The Minnesota State Fire Marshall's Office was recently on campus to film a Campus Fire Safety video that will be used as a resource for all Minnesota colleges and universities. Assisting with the video were Gustie students Summer Boettcher, Audi Dickey, Paget Pengelly and McCartney Renn. A dormitory fire drill was also captured in the footage as all the dorms practiced their evacuation procedures as part of State-wide and National Campus Fire Safety Month activities.
Campus fire safety is a serious issue. According to the National Fire Protection Association, more than 3,800 university housing fires occur each year in the United States, causing 5 deaths, 50 injuries and $26 million in property loss. There are various causes of campus fires, but the fundamental issue is general lack of knowledge about fire safety and prevention. Cooking is the leading cause of fires (over 83%), followed by arson, open flame, heating, and unintentional or careless behavior. Students can prepare for a safe new life away from home by following a few simple guidelines:
- When cooking, stay in the kitchen area and pay attention to the cooking process. Leaving food cooking unattended, for even a minute, can be deadly. If a pan starts on fire, cover it with a lid and turn off the stove. Never throw water on a grease fire as it will spread the fire.
- Never ignore fire alarms! Do not wait to see fire or smoke. If your keys and cell phone are close by, take them with as you evacuate and close your door behind you. Know the exit routes from your building and the designated evacuation area. Never go back into a building that is being evacuated. Your life is more important than your stuff!
- Campus housing is equipped with smoke and carbon monoxide alarms and fire suppression sprinklers that are inspected routinely. If you live off-campus, inspect your smoke and carbon monoxide detector and make sure the battery is changed at least once a year. This little alarm can save your life.
- Do not use candles or incense as they are a fire danger. Consider flameless candles that look and smell like real candles. Avoid overloading wall outlets and using extension cords. Consider use of a power strip.
- Smoking is the leading cause of fire fatalities and not allowed in Campus Housing. If off-campus, never smoke if too tired or impaired. Always put your cigarette out completely. Cigarettes can smolder for hours and start a fire long after you go to sleep.
- Alcohol consumption is often a factor in campus fire fatalities. Alcohol can impair judgment and hamper evacuation efforts. Assign an "event monitor" who stays unimpaired.
|Jul 9 5:30–7:30 pm
|Summer Gatherings - Crosslakehttps://gustavus.edu/calendar/summer-gatherings-crosslake/45184Crosslake, MN
|Jul 11–
|Summer Gatherings - 1995 Gustavus Choir Tourhttps://gustavus.edu/calendar/summer-gatherings-1995-gustavus-choir-tour/45389Twin Cities
|Jul 11–
|Summer Gatherings - Twin Cities Weekhttps://gustavus.edu/calendar/summer-gatherings-twin-cities-week/45016Twin Cities
|Jul 11 9 am to 6 pm
|Twin Cities Week - Minnesota Zoohttps://gustavus.edu/calendar/twin-cities-week-minnesota-zoo/45022Minnesota Zoo 13000 Zoo Blvd, Apple Valley, MN 55124
|7–9 pm
|Twin Cities Week - Minnesota Unitedhttps://gustavus.edu/calendar/twin-cities-week-minnesota-united/45023National Sports Center Stadium 1700 105th Ave NE, Blaine, MN
|Jul 12 All day
|Twin Cities Week - Gustavus Sabbathhttps://gustavus.edu/calendar/twin-cities-week-gustavus-sabbath/45086Across the country
|Jul 13 All day
|Twin Cities Week - Date Nighthttps://gustavus.edu/calendar/twin-cities-week-date-night/45024Multiple Locations
|Jul 14 9–11 am
|Kirk Beyer and Nadine Haukoos Retirement Receptionhttps://gustavus.edu/calendar/kirk-beyer-and-nadine-haukoos-retirement-reception-2/45624Heritage Room
|6–9 pm
|Twin Cities Week - Maynardshttps://gustavus.edu/calendar/twin-cities-week-maynards/45025Maynards 685 Excelsior Blvd., MN 55331
|Jul 15 6–9 pm
|Twin Cities Week - Gusties Take the Stagehttps://gustavus.edu/calendar/twin-cities-week-gusties-take-the-stage/45615American Swedish Institute, 2600 Park Avenue, Minneapolis, MN 55407
|Jul 16 10 am to 2 pm
|Twin Cities Week - Golf Outinghttps://gustavus.edu/calendar/twin-cities-week-golf-outing/45610Legends Club - Prior Lake, MN
|noon to 1 pm
|HERC Webinar: "Demystifying the H-1B Visa Process: What Higher Education Supervisors Need to Know"https://gustavus.edu/calendar/herc-webinar-demystifying-the-h-1b-visa-process-what-higher-education-supervisors-need-to-know/44089Beck Hall 121
|2–3:30 pm
|Retirement Party for Dean Wahlund and Steve Waldhauserhttps://gustavus.edu/calendar/retirement-party-for-dean-wahlund-and-steve-waldhauser-2/45614Alumni Hall
|5–9 pm
|Twin Cities Week - Art Museumhttps://gustavus.edu/calendar/twin-cities-week-art-museum/45027Walker Art Center 1750 Hennepin Ave, Minneapolis, MN 55403
|Jul 17 All day
|Summer Snapshot - Admission Visit Event for Prospective Students and Familieshttps://gustavus.edu/calendar/summer-snapshot-admission-visit-event-for-prospective-students-and-families-2/45594Alumni Hall, Student Union and Campus Center
|7:30–10 pm
|Twin Cities Week - The Music Manhttps://gustavus.edu/calendar/twin-cities-week-the-music-man/45049Guthrie Theater 818 South 2nd Street, Minneapolis, MN 55415
To add or change items on the calendar, please fill out and submit a College Calendar event form. View the entire College Calendar online.
Where Nice Evenings Are Recalled by Kurt Elling '89
2 weeks ago by Al Behrends
From Fine Arts
Elizabeth Weiers Named Capital One Academic All-American
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From Athletics » Featured Homepage
Hillstrom Museum of Art Features Summer Viewing of Two Sanzén Landscapes
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Katie Sexe-Talbot '00: A Leader in Healthcare Administration
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Looking Ahead to the 51st Nobel Conference
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From News » Third
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Carolyn Draayer And Katie Olson Named Capital One Academic All-Americans
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Video and Photos from Commencement Day
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David Lilly and Jenny Strom Named Gustavus Student-Athletes Of The Year
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Symphony Finale Features Aria/Concerto Winners
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From Fine Arts
Senior Art Majors Exhibition Opens May 2 in Hillstrom Museum
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Coming Weekend Concludes Gustavus Recital Season
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Inside Gustavus is a newsletter for Gustavus Adolphus College employees produced by the Office of Marketing and Communication. It is published weekly during the academic year (except during the week of Thanksgiving, the Christmas break, Touring Week, and the Spring and Easter breaks). Anyone may submit items by filling out an online submission form. While online submissions are preferred, items may also be submitted typewritten on a letter-sized sheet of paper. Send "snail mail" items to: Inside Gustavus, Office of Marketing and Communication. Items must reach the office no later than 4:30 p.m. on the Tuesday before publication. For more information, contact Matt Thomas (firstname.lastname@example.com or x7510).
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