text:
Expressed throughout the central nervous system, the myocardin-related, megakaryoblastic acute leukemia 1 and 2 (Mkl1/2) are transcriptional cofactors that can be found tethered in the cytoplasm to monomeric actin but on synaptic activation translocate to the nucleus and associate with transcription factors such as serum response factor (SRF) to regulate expression of structural genes. This implies a potential role for Mkls in linking synaptic activity, through gene-expression control, to neuronal structural plasticity. Here, we present evidence that Mkls, particularly Mkl2, are powerful regulators of neuronal structure in vitro. Moreover, using the passive avoidance–conditioning paradigm, we identify learning-associated alterations of neuronal Mkl expression that appear to contribute to 2 phases of gene regulation during memory consolidation in the hippocampus. Gene regulation immediately after learning includes Egr2 and may be facilitated by downregulation of Mkls likely releasing ternary complex factor–regulated SRF activity. The second transcriptional phase occurs later at the 3-h postavoidance time point when Mkl accumulates in the nucleus of hippocampal neurons and there is enhanced transcription of Mkl-dependent structural genes that may contribute to the elaboration of new, memory-associated synapses known to appear over the subsequent 3-h period.
Serum response factor (SRF) is a highly conserved transcription factor that regulates the expression of a host of stimulus-dependent genes. Within the central nervous system (CNS), the specific repertoire of genes induced by a particular stimulus is largely dictated by the interaction of SRF with 1 of 2 families of cofactors that compete for an overlapping binding site on SRF (Gineitis and Treisman 2001; Zaromytidou et al. 2006). The first of these, the relatively well-studied ternary complex factor (TCF) family, has been shown to mediate the expression of many immediate early genes (IEGs) (Sgambato et al. 1998; Cesari et al. 2004). The second family is the myocardin-related cofactors, of which megakaryoblastic acute leukemia 1 (Mkl1; also known as MAL1 and MRTF-A) and Mkl2 are expressed throughout the CNS (Wang et al. 2002; Miralles et al. 2003; Shiota et al. 2006). In vitro studies have shown that Mkl proteins bind to monomeric actin in the cytoplasm via 3 RPEL motifs (RPXXXEL; Guettler et al. 2008). Increased Rho-activated actin polymerization causes Mkl proteins to accumulate in the nucleus (Vartiainen et al. 2007), where they bind to SRF and mediate the expression of a large number of genes with functions related to actin dynamics (Posern and Treisman 2006; Miano et al. 2007).
To date, studies investigating the function of Mkl proteins within the CNS have focused on Mkl1. Mkl1 has been found to translocate to the nucleus of cultured cortical neurons following Rho-A signaling, synaptic activation, and membrane depolarization implying a potential role for this transcriptional cofactor in linking synaptic activity, through gene expression control, to neuronal structural plasticity (Tabuchi et al. 2005; Kalita et al. 2006). However, there exists some discrepancy in the literature as to the subcellular localization of Mkl1 within neurons: Some groups report that it is largely cytoplasmic and others constitutively nuclear (Kalita et al. 2006; Shiota et al. 2006; Stern et al. 2009). It is unclear if this represents differences in cell populations or differences between the antibodies used in these immunohistochemical studies. Moreover, in vitro work has shown that there is functional redundancy between Mkl1 and Mkl2, with complete blockade of serum response element–dependent reporter gene activity requiring inhibition of both Mkl1 and Mkl2 (Cen et al. 2003; Li et al. 2006). Despite this, to our knowledge, the role of Mkl2 within neurons has not been investigated.
Tightly regulated synaptic plasticity and physical reorganization of neuronal connections, particularly within the hippocampus, is critical to consolidation of long-term memory (LTM) (O'Malley et al. 1998, 2000; Eyre et al. 2003; Morris et al. 2003; Lamprecht and LeDoux 2004). Although there has been much debate as to the nature of the plasticity, it is now clear that both functional (synapse strength) and morphological (synapse size and position) plasticity occur (Bliss and Collingridge 1993; Malenka and Nicoll 1999; Geinisman 2000). At the cellular level, LTM is dependent on de novo gene and protein expression and is sensitive to inhibitors of transcription and translation (Davis and Squire 1984; Quevedo et al. 1999; Igaz et al. 2002). Such regulation of gene expression occurs in coordinated waves during memory consolidation, controlled by transcription factor activity, providing the raw materials for the long-term neuronal changes needed for information storage (Igaz et al. 2002). Moreover, SRF is known to be one of the transcription factors involved in regulating hippocampal plasticity (Dash et al. 2005; Ramanan et al. 2005; Miano et al. 2007).
To investigate the function of Mkl1/2 proteins in hippocampal neurons, we carried out individual and combined knockdown of expression using small interfering RNAs (siRNAs). We found that dysregulation of Mkl1/2 expression inhibits Mkl SRF–dependent gene expression and disrupts neurite growth and branching. In addition, we explored the role of endogenous Mkl1/2-dependent gene regulation in discrete subregions of the adult hippocampus following passive avoidance training. These results lead us to suggest that Mkl1/2 competes for SRF binding in hippocampal neurons to regulate neuronal structural plasticity during memory formation.
Materials and Methods
Animal Maintenance and Training
Postnatal day 80 male Wistar rats (330–350 g) were individually housed on a 12:12 light/dark cycle, with ad libitum access to food and water. Animals were assessed for 3 days prior to passive avoidance training by observing their open field behavior. The 1-trial, step-through, light–dark version of the passive avoidance paradigm was employed as we have described previously (Fox et al. 1995). The smaller illuminated compartment was separated from the larger dark compartment by a shutter that contained a small entrance. The floor of the training apparatus consisted of a grid of stainless steel bars that would deliver a scrambled shock (0·75 mA every 0·5 ms) for 5 s when the animal entered the dark chamber. Passive controls were exposed to the avoidance apparatus for a time matched to trained counterparts but never received a foot shock. Animals were killed by cervical dislocation at 0 and 3 h following training (4 animals per group, 16 animals in total). For immunohistochemical analysis, brains were quickly dissected out, covered in optimal cutting temperature compound and snap frozen in CO2-cooled n-hexane. For real-time polymerase chain reaction (PCR), the dentate gyrus (DG) was rapidly dissected, snap frozen in liquid nitrogen, and stored at −80 °C until required. All experimental procedures were approved by the Animal Research Ethics Committee, University College Dublin and were carried out by individuals who held the appropriate license issued by the Irish Minister for Health and Children.
Primary Neuronal Culture
Rats at embryonic day 17 were used to prepare low-density primary hippocampal neuronal cultures. After complete removal of meninges, hippocampi were dissected in Hank's balanced salt solution (HBSS). The tissue was triturated in HBSS with 0.05% trypsin and dissociated by repeated pipetting. Cells were plated in cortical plating medium (neurobasal medium (NB), 2% B27 + supplement, 0.5-mM L-glutamine, penicillin, and streptomycin) on poly-D-lysine coated coverslips in 12-well plates at a density of 1.5 × 104 cells per well (low density) or 1.5 × 105 cells per well (high density). Plated neurons were maintained in an incubator with 5% CO2 at 37 °C. Staining with the neuron-specific marker NeuN indicates that plating at a low density produces very pure neuronal cultures (Fig. 1E), whereas plating at the higher density results in a more heterogenous mix of neuronal and nonneuronal cells (Fig. 3A).
siRNA Knockdown
Synthetic siRNAs targeting rat Mkl1 (L-081405-00) and Mkl2 (L-103684-01), as well as a nontargeting control (NTC; D-001810-10), were obtained from Dharmacon Inc. (Thermo Scientific, Northumberland, United Kingdom). Twenty-four hours after hippocampal neurons were plated on coverslips, as described above, the medium was replaced with antibiotic-free maintenance medium (NB, 2% B27 minus supplement, 0.5 mM L-glutamine) containing 100 nM siRNA: 100 nM of NTC, Mkl1 siRNA, Mkl2 siRNA, or 50 nM each Mkl1 and Mkl2 siRNA for double knockdown. Transfection was achieved using DharmaFECT 3 reagent following the manufacturer's instructions. All experiments were performed in triplicate and repeated with at least 3 different platings.
Real-Time PCR
Total RNA was purified from individual wells or DG in TRIzol reagent (Invitrogen, United Kingdom) according to the manufacturer's instructions. cDNA was produced using SuperScript II RNase H Reverse Transcriptase Kit (Invitrogen, United Kingdom) using 50–250 ng of random primers. Real-time PCR was carried out using TaqMan technology on an ABI Prism 7900HT sequence detection system (PE Applied Biosystems, United Kingdom). The mRNA expression for Mkl1, Snai2, tropomyosin 3 (Tpm3), alpha smooth muscle actin (α-SMA), and Egr2 was determined using TaqMan Gene Expression Assay primers and probes (Applied BiosystemsAssay ID Rn01458644_m1, Rn00709370_m1, Rn00589483_m1, Rn01759928_g1, and Rn0058622_m1, respectively. Assayed primers and probes were not available for Mkl2 so were designed as follows: forward-GACCTCATTGAACGCGTGAAA, reverse-ATACTGCCAGTGGCAAGGTTATTAC, and probe-CCTACCAGGAAGTGACC. The mRNA expression was quantified by constructing a standard curve for the primer and probe set with pooled DNA from all samples. A ribosomal RNA control primer and probe set served as an internal control (Applied Biosystems).
Protein lysates from high-density plates treated for 4 days with siRNA were separated on polyacrylamide minigels and electrophoretically transferred to nitrocellulose membranes (Bio-rad). Equal protein loading was confirmed by ponceau S staining of the membrane (not shown). The nitrocellulose was blocked in 5% nonfat milk in 10 mM Tris–HCl, 150 nM NaCl, and 0.05% (vol/vol) Tween-20 (Tris-buffered saline tween-20 [TBS-T]) for 1 h at room temperature. Rabbit anti-Mkl1/2 (Sc-32909; Santa Cruz, CA) was diluted in 5% milk and incubated overnight at 4 °C. After incubation with antirabbit horseradish peroxidise (A6154, Sigma) and SuperSignal Chemiluminescent Substrate (Pierce), bands were visualized by exposure to X-ray film (Kodak).
Structural Analysis
Cells were grown in siRNA-treated medium for 4 days, which allowed for growth of neurites while remaining distinct from surrounding neurons. Coverslips were blinded before the cells were fixed in 70% ethanol for 30 min and images captured by phase contrast microscopy. Neuronal morphology was analyzed using Sholl analysis, which measures the density of neurites as a function of distance from the cell body (Sholl 1953). In this study, concentric digitally generated rings 4.9 μm apart were centered on the cell soma, and the neurites intersecting each ring were counted. Total length of neurites was determined by automated analysis, whereas branch point numbers were manually counted.
Whole brains were cryosectioned horizontally at −7.1 mm with respect to Bregma to reveal the ventral hippocampus. Twelve-micrometer sections were adhered to glass slides coated with poly-L-lysine. One section from each rat brain was used for analysis, and each trained section was prepared, stained, and imaged along with their relevant passive control.
Immunocytochemistry and Confocal Microscopy
Coverslips or sections were fixed in 70% ethanol, permeablized using a solution of 0.2% Triton X in phosphate buffered saline (PBS), and incubated overnight (18 h) with primary antibody. We used rabbit anti-Mkl1/2 (Sc-32909, Santa Cruz), mouse anti-NeuN (MAB377, Chemicon), and mouse anti-glial fibrillary acidic protein (GFAP; Ab10062, Abcam). After washing with PBS, cells or sections were incubated with the appropriate Cy3− or fluorescein (FITC)-conjugated fluorescent antibodies. The secondary antibody was then washed off, and samples were incubated with either propidium iodide (PI) or Hoechst, in order to stain all nuclei. If not imaged immediately, sections were mounted with Citifluor (glycerol in PBS) and stored in darkness at 4 °C.
All confocal images (1024 × 1024 pixels, 0.22-μm × 0.22-μm pixel size, and 3.6-μm optical slice) used for analysis were captured using identical settings. Images taken from hippocampal sections were captured at 4 distinct regions of the hippocampus, that is, CA1, CA2, CA3, and the apex of the DG. These specific areas of the hippocampal neuronal circuit were kept consistent between sections (Fig. 4A).
Image Analysis
Image analysis was conducted using the Bioconductor EBImage plugin for R (Sklyar et al. 2009), which has an implementation of the watershed segmentation algorithm, as this approach to image analysis allows the automated and accurate separation and fluorescence intensity measurement in individual cells that are very close together or touching. Using watershed segmentation, the nucleus of each cell was defined from a binary threshold image derived from the red channel of each image, corresponding to the PI iodide staining. Threshold levels, distance between objects, and minimum object radii were set for each region independently to select only nuclei. After nuclear identification, green channel intensity, which corresponds to Mkl1/2 expression, was quantified for each nuclear region. To assess expression in a cytoplasmic region around each nucleus, the nuclear threshold image was "dilated" using morphological kernel expansion, and image segmentation repeated, beginning at the identified nuclei. This resulted in a "whole-cell" object being defined corresponding to each nucleus, in which green channel fluorescence was quantified. Cytoplasmic fluorescence levels were calculated by subtracting nuclear from whole-cell values, and nuclear:cytoplasmic ratios were then calculated according to the equation [(Fn – Fc)/Fc], where Fn = average nuclear fluorescence and Fc = average cytoplasmic fluorescence.
For siRNA experiments, protein levels were assessed on a cell-by-cell basis by immunocytochemistry and analyzed using the same image segmentation and intensity measurement approach. In this case, mean intensity across the whole cell, as opposed to nuclear/cytoplasmic intensity ratio, was used as an index of protein levels in each cell.
Statistical Analysis
Statistical significance was calculated using 1 way analysis of variance (ANOVA) and Dunnett's post-hoc tests (P < 0.01), 2way ANOVA, and Bonferonni post-hoc tests (P < 0.01), or by Student's t-test analysis (P < 0.05), as appropriate.
Statistical significance of Mkl1/2 localization changes was determined using a Kolmogorov–Smirnov (KS) test, performed on the data using R (P < 0.01). Normal parametric statistical tests, such as Student's t-test, can only detect changes in a population mean and is therefore most useful in detecting more homogeneous changes in a population. However, the automated process of gathering data in this study resulted in the measurement of nuclear:cytoplasmic ratios in ∼125–250 cells in each hippocampal subregion in each sample (4 samples in each trained/passive group; total cell count for each subregion available in table S1). This relatively large sample size allows the use of a nonparametric statistical test that can detect any changes in a population. In contrast to Student's t-test, the KS test has the advantage of making no assumption about the distribution of data and so can detect differences between populations even if there is marked heterogeneity in the response of the cell population to a learning event. The D statistic, the maximum deviation between the populations, was also generated.
Knockdown of Mkl1 in Hippocampal Neurons Can Result in a Concomitant Upregulation of Mkl2 Expression
We knocked down expression of Mkl genes in hippocampal neurons by transfecting 100 nM siRNAs for Mkl1 and Mkl2. Following treatment with Mkl1 siRNA for 48 and 96 h, we see effective reduction of Mkl1 mRNA from controls (50 ± 7% and 56 ± 7% of control, respectively, 1-way ANOVA and Dunnett's post-hoc test, P < 0.01; Fig. 1A,C). However, Mkl1 siRNA treatment also produced an increase in Mkl2 mRNA from controls (159 ± 11% and 148 ± 20% from control for 48 and 96 h, respectively, P < 0.01 at 48 h P > 0.05 at 96 h; Fig. 1B,D). Forty eight– and 96-hour treatment with Mkl2 siRNA results in reduced Mkl2 mRNA levels (44 ± 5% and 49 ± 15% of control, respectively, P < 0.01 at 48 h and P < 0.05 at 96 h; Fig. 1B,D) with no change in Mkl1 expression observed. Finally, we simultaneously knocked down Mkl1 and Mkl2, using 50 nM of each siRNA, which resulted in decreased expression of Mkl1 and Mkl2 at 48 h (43 ± 5% and 41 ± 6% of control, respectively, P < 0.01; Fig. 1A,B) although this was less robust as it was not maintained at 96 h (80 ± 7% and 84 ± 15% of control, respectively, P > 0.05; Fig. 1C,D). One-hundred nanomoles of NTC siRNA showed no significant change from untransfected control levels in any parameter investigated.
We immunostained siRNA-treated neurons with an antibody purporting to be reactive to Mkl1 (Sc-32909; Santa Cruz). We did not see any change in protein intensity 48 h following treatment (data not shown). However, 96 h posttreatment we found decreased antibody staining in cells treated with Mkl1 siRNA, Mkl2 siRNA, and the double knockdown (80 ± 2%, 87 ± 2%, and 84 ± 2% of control respectively, P < 0.01; Fig. 1E,F). This provides evidence that this antibody binds both Mkl1 and Mkl2. Moreover, immunoblotting of protein lysate from neurons 96 h following siRNA treatment reveals a decrease in a ∼98-kDa band, the expected molecular weight of rat Mkl1 (Fig. 1G).
Altered Mkl1/2 Expression in Hippocampal Neurons Modulates Neuritic Growth and Branching
We observed that treatment with Mkl1 and Mkl2 siRNAs resulted in significant changes to neuronal structure (Fig. 2A). This effect was not immediate (no structural changes seen at 48 posttreatment, data not shown) but was visible 96 h posttreatment, coincident with the alteration in Mkl1/2 protein level. Specifically, neurons treated with Mkl1 siRNA, which have decreased Mkl1 and increased Mkl2 expression, have significantly more neurite complexity (assessed by Sholl distribution) compared with controls (2-way ANOVA and Bonferonni post-hoc tests, P < 0.01; Fig. 2B and Fig. S1A). In addition, these neurons had increased neurite branching (24 ± 1 compared with 18 ± 1 branch points for controls, P < 0.01; Fig. 2C), total neuritic length (1348 ± 58μm compared with 826 ± 27 μm for controls, P < 0.01; Fig. 2D), and longer maximal neurite distance from soma (210 ± 12 μm compared with 124 ± 5 μm for controls, P < 0.01; Fig. S1B). In contrast, neurons treated with Mkl2 siRNA, which have decreased Mkl2 but normal Mkl1 expression, and the double knockdown, which have decreased Mkl1 and Mkl2 expression, have significantly less Sholl neurite complexity compared with controls (P < 0.01; Fig. 2B and Fig. S1A). These neurons also have decreased branching (10 ± 1 and 8 ± 1, respectively, P < 0.01; Fig. 2C), total neuritic length (508 ± 23 and 443±26 μm, respectively, P < 0.01; Fig. 2D), and longer maximal neurite distance from soma (87 ± 4 and 77 ± 4 μm, respectively, P < 0.01; Fig. S1B) compared with controls.
We next explored if these structural changes were associated with altered expression of Mkl1/2 target genes as a result of the siRNA treatment. Coincident with the change in protein level and altered neuritic structure at 96 h posttraining we found decreased expression of the SRF Mkl–regulated genes αSMA and Tpm3. Specifically, αSMA expression was decreased in Mkl2 siRNA and double knockdown–treated cells (27 ± 4% and 47 ± 9% of control, respectively, P < 0.01; Fig. 2E). Tpm3 expression was significantly decreased in Mkl1 siRNA and double knockdown–treated cells (76 ± 4% and 74 ± 7% of control, respectively, P < 0.05) and decreased in Mkl2 siRNA–treated cells, though this did not reach significance (78 ± 4% of control, P > 0.05; Fig. 2F). In contrast, expression of the Mkl–SMAD3 regulated snail family transcription factor member Snai2 was not altered by the siRNA treatments (Fig. 2G).
Mkl1/2 Is Expressed in Hippocampal Neurons and Astrocytes In Vitro and In Vivo
There has been debate in the literature as to the cellular localization of Mkl1/2 protein in CNS tissue. As this point is fundamental to an understanding of the role of Mkl1/2 in neuronal function, we next established where Mkl1/2 was expressed across the major subtypes of hippocampal cells. In primary hippocampal cultures, colocalization of Mkl1/2 with both the neuronal marker NeuN and the actrocytic marker GFAP indicated that Mkl1/2 was expressed in both neurons and astrocytes (Fig. 3A and Fig. S2). In addition, Mkl1/2 was clearly expressed in both the nucleus and cytoplasm of neuronal cells (Fig. 3A and enlarged in Fig. S2A, arrowheads).
Immunohistochemical analysis of hippocampal slices from adult rat brain revealed widespread colocalization of Mkl1/2 staining with NeuN across the hippocampus (Fig. 3B and Fig. S3). There was also some evidence for Mkl1/2 colocalization with GFAP staining in the intact adult hippocampus (Fig. 3B and Fig. S4, arrow), though only a small proportion of GFAP-positive cells exhibited appreciable Mkl1/2 expression.
Mkl1/2 Undergoes Learning-Specific Nuclear Accumulation in Hippocampal Neurons 3 h Following Passive Avoidance Training
As the studies presented above indicate that Mkl1/2 can be detected and quantified in the primary excitatory neurons in the subregions of the adult hippocampus, we wanted to determine whether it undergoes nuclear translocation following a learning event. Equivalent horizontal sections of the ventral hippocampus (−7.1 mm w.r.t. Bregma) were taken from animals 0 and 3 h following passive avoidance training and from corresponding passive controls. High-magnification confocal images were taken of Mkl1/2 staining in the granule cells of the DG and the pyramidal cells of the CA regions of the ventral hippocampus (CA1-3; Fig. 4A). Double labeling using Mkl1/2 and the nuclear marker PI demonstrates that Mkl1/2 protein can be detected in the nucleus and somatic cytoplasm of cells in all 4 hippocampal subregions (Fig. 4B). Using a cell-by-cell analysis approach, the relative amount of Mkl1/2 present in different cellular compartments, that is, nucleus and somatic cytoplasm, within each distinct cell population of the hippocampus was quantified. Interestingly, though detectable in both compartments, the levels of Mkl1/2 expression are consistently greater in the nucleus than in the cytoplasm (Figs. S5 and S6). Finally, the nuclear:cytoplasmic ratio for each cell was calculated. Within each subregion, these ratios were pooled and used for comparison between trained and passive samples at both posttraining time points.
Given that there is no compelling a priori reason to believe that every cell in a given hippocampal region would respond in the same way to a learning stimulus, it is important to select an analytical approach that allows the detection of heterogenous changes after learning. Indeed, we observed that Mkl1/2 levels are not uniform across the hippocampus, being highest in the CA3 region and lowest in the DG (Figs. S5 and S6). When the nuclear:cytoplasmic ratios from the hippocampal regions were analyzed by t-tests, differences were only detected in the CA2 region 3 h posttraining (Fig. S7). However, parametric statistics based on a normal distribution are particularly insensitive to differences in skew or kurtosis of 2 distributions, which is an important source of variation if a population responds to a stimulus in a heterogeneous manner. As such, the trained and passive control values in each subregion were compared using the KS test and the differences between populations illustrated graphically using cumulative frequency distributions. In these frequency distributions, a rightward shift of a distribution indicated a relatively higher nuclear expression, and, conversely, a leftward shift indicated relatively less accumulation within the nucleus in that part of the distribution.
No change in Mkl1/2 cellular distribution was detected immediately following training, in any of the hippocampus cell populations studied (Fig. 4C; D = 0.0.3, P = 0.76 for DG; D = 0.08, P = 0.014 for CA3; D = 0.07, P = 0.1 for CA2; and D = 0.05, P = 0.35 for CA1). In contrast, we found that the nuclear:cytoplasmic ratio of Mkl1/2 was consistently higher in trained animals relative to passive controls for all hippocampal regions studied 3 h following training (Fig. 4C; D = 0.15, P = 2.4 × 10−10 for DG, D = 0.12, P = 1.2 × 10−4 for CA3, D = 0.15, P = 5.8 × 10−6 for CA2, and D = 0.09, P = 6.8 × 10−3 for CA1). These findings indicated that nuclear accumulation of Mkl1/2 occurs across the hippocampal subregions at this time following avoidance conditioning.
Upregulation of Mkl1/2-Induced Genes Snai2, Tpm3, and α-SMA in the Hippocampal DG Coincident with Increased Mkl1/2 Nuclear Localization
Having shown that Mkl1/2 accumulated within the nucleus of hippocampal neurons 3 h postlearning, we wanted to investigate if this contributes to memory-associated gene regulation. The expression readout of 3 genes whose transcription is known to be regulated by Mkl1/2 was used to see if transcription of Mkl1/2 target genes increased coincident with the observed nuclear translocation. The DG, a region established to undergo synaptic reorganization during consolidation (O'Malley et al. 1998, 2000), was dissected from trained and passive control animals 0 and 3 h following training. mRNA was isolated from these samples and real-time PCR analysis carried out. We detected significantly increased expression of the Mkl–SRF activated genes Tpm3 and α-SMA 3h, but not 0 h, following passive avoidance training (150 ± 16% and 160 ± 15% of control respectively, P < 0.05; Fig. 5). We also found significantly increased expression of the Mkl–SMAD3 activated gene Snai2 3h and not 0 h posttraining (204 ± 29% of control, P <0.05; Fig. 5).
In contrast, the TCF–SRF activated gene Egr2, whose expression has been shown to be independent of Mkl1/2 signaling (Selvaraj and Prywes 2004), did not increase at the 3 h time point. In keeping with what is known about the regulation of IEGs following learning, however, we observed increased Egr2 expression 0 h posttraining (140 ± 18% of control, P < 0.05; Fig. 5). In addition, we found that the expression levels of Mkl1 and Mkl2 were not themselves increased at the 3 h time point but, interestingly, were decreased at the 0 h time (75 ± 9% and 55 ± 10% of control, respectively, P < 0.05; Fig. 5).
Regulation of Hippocampal Neurite Structure —a Dominant Role for Mkl2
Previous studies have suggested a role for Mkl1 in neuronal structural control. For example, transfection with Mkl1 siRNA–expressing plasmids in high-density cortical cultures results in reduced neuritic growth, whereas neurite outgrowth in cortical and dorsal root ganglia cells is disrupted in dominant negative (DN) mutants of Mkl1 (Shiota et al. 2006; Wickramasinghe et al. 2008). One of the key findings of the present study is the dominate effect that Mkl2 expression has on control of neuritic growth in hippocampal neurons. We found that increased Mkl2 was associated with enhanced neurite outgrowth and branching, whereas decreased Mkl2 significantly inhibits neuritic growth. Interestingly, the previous studies on Mkl1 function did not investigate the effect of the siRNA plasmid or DN mutation on Mkl2 expression, and so we cannot eliminate the possibility that Mkl2 expression was also affected and contributed to the structural deficits observed in those studies. In support of this suggestion, DN Mkl2 mutants have been shown to block activation of both Mkl1 and Mkl2 (Selvaraj and Prywes 2003) raising the possibility that a similar effect occurs for the Mkl1 DN.
Subcellular Localization of Mkl1/2 in Hippocampal Neurons
As mentioned previously, no consensus has been reached as to the subcellular localization of Mkl1/2 (Kalita et al. 2006; Shiota et al. 2006; Stern et al. 2009). Importantly, the Mkl1 antibody used in the present study clearly detects both Mkl1 and Mkl2 proteins. As such, studies using this antibody should refer to Mkl1/2 and not Mkl1 alone. Using this antibody, we report that Mkl1/2 is endogenously expressed in both the cytoplasm and the nucleus of hippocampal neurons in culture and in adult brain. It is noteworthy that our analysis reveals nuclear expression to be more substantial than cytoplasmic in hippocampal neurons (Figs. S6 and S7) and that the Mkl1/2 outside of the nucleus is largely detected in the somatic cytoplasm. Clearly, other antibodies used previously may simply be more specific for Mlk1 and correctly identify this protein to be restricted to the nucleus. This would imply that the cytoplasmic expression seen with our antibody is attributable to Mkl2.
Hippocampal Nuclear Regulation of Mkl1/2 Following Passive Avoidance Learning
Mkl1/2 shuttles between the nucleus and the cytoplasm, even in unstimulated cells (Vartiainen et al. 2007) and has been found to translocate to the nucleus of cortical cells in response to activation of Rho-A signaling (Tabuchi et al. 2005). Here, we provide evidence for dynamic movements of Mkl1/2 between cytoplasmic and nuclear compartments following a learning event. Specifically, Mkl1/2 translocates to, and accumulates within, the nucleus of granule and pyramidal neurons 3 h following passive avoidance training. The functional relevance to learning of such Mkl1/2 nuclear accumulation likely relates to the coincident increased expression of the Mkl1/2-dependent genes, Tpm3, αSMA, and Snai2 in the hippocampal DG. Expression levels of Tpm3 and αSMA are regulated by Mkl1/2-SRF, with αSMA expression in Mkl1 knockout mice attributed specifically to Mkl2 (Li et al. 2006). Our current findings support a direct regulatory role as knockdown of Mkl1 and Mkl2 inhibits transcription of these genes with a particularly profound reduction in αSMA seen following treatment with Mkl2 siRNA. Snai2, a negative regulator of E-cadherin expression, is instead controlled by the Mkl–SMAD3 transcription complex (Schmidt et al. 2005; Morita et al. 2007). Snai2 levels were also increased in the DG 3 h posttraining, but were not affected by dysregulation of Mkl1 or Mkl2 expression in vitro suggesting that basal expression of this gene is Mkl independent, whereas learning-associated activation may still be driven by these transcription cofactors.
Altogether, Mkl targets comprise an impressive list of genes with functions related to actin-dynamics (Miano et al. 2007). The bidirectionality of the relationship between actin dynamics and Mkl signaling can be seen with actin mutations that inhibit actin polymerization, reducing neurite length and downregulating levels of the reported full-length Mkl1/2 protein (Stern et al. 2009). These properties of Mkl signaling, coupled with the postlearning time of regulation preceding the appearance of new synapses (O'Malley et al. 1998, 2000; Eyre et al. 2003), identify Mkl as an ideal candidate to drive transcriptional events underpinning memory-associated synaptic reorganization.
Synaptic activity, via the MAPK/ERK kinase pathway, activates TCF–SRF mediated transcription of IEG expression (Xia et al. 1996; Gokce et al. 2009). This pathway is crucial for the initiation of a cascade of downstream genes required to mediate long-term synaptic plasticity. Here, we confirm the upregulation of the IEG Egr2 0 h posttraining. Strikingly, Egr2 expression is controlled by TCF-SRF activity independent of Mkl1/2 (Selvaraj and Prywes 2004). Also, at 0 h, gene expression and corresponding protein levels of Mkl1/2 are significantly decreased, and there is no active shuttling of Mkl1/2 between subcellular compartments. Given that Mkl1/2 and TCF compete for an overlapping binding site on SRF (Zaromytidou et al. 2006), it is tempting to suggest that decreased Mkl1/2 expression immediately following training in the hippocampus may facilitate TCF SRF–mediated IEG expression necessary at this time (Fig. 6). This theory is supported by studies showing that the TCF Elk-1 can dissociate Mkl1/2 from SRF and that expression levels of Mkl–SRF and TCF–SRF genes are inversely correlated (Yoshida et al. 2007; Descot et al. 2009).
These studies have characterized a role for Mkl transcription cofactors, particularly Mkl2, in regulation of neuronal structure both in vitro and in vivo. These findings support a model where regulation of Mkl1/2 nuclear accumulation contributes to 2 waves of gene regulation during memory consolidation (Fig. 6). The first, immediate gene regulation relates to IEGs may be facilitated by downregulation of Mkls releasing TCF-SRF activity. The second transcriptional wave occurs later at the 3 h postavoidance time point when Mkl accumulates in the nucleus of hippocampal neurons enhancing transcription of Mkl-dependent structural genes that may contribute to the elaboration of new, memory-associated synapses that appear over the subsequent 3-h period.
Supplementary Material
Supplementary material can be found at: http://www.cercor.oxfordjournals.org.
Conflict of Interest: None declared.


text:
With Instructables you can share what you make with the world, and tap into an ever-growing community of creative experts.
my entry for T.N.K.I.T i know it sukz but (a) it is my first car and (b) i am not the worlds best knex builder so if i ...
Join 2 million + to receive instant DIY inspiration in your inbox.
© 2016 Autodesk, Inc.

subset: commoncrawl


text:
ABRAMS, Wis. (WBAY) – It was like Christmas morning for all the hunters in Wisconsin because Saturday marked the opener for the gun deer hunting season.
Plenty of hunters stopped at a BP gas station in Abrams, picking up items to get themselves through the long hours in the deer stands.
Hunters have been anticipating getting out to the woods for weeks. They got out their deer stands and made sure their guns were ready to go.
"We are acting crazy and going to look in the snow for brown, furry things with white tails," said hunter Mike Rodgers.
"This is opening day of gun season, and [I'm] hoping to get some venison in the freezer," said hunter Billy Patterson.
Most importantly, hunters got all of their winter gear out of the closet. If you ask any hunter, there's no telling how long you will be outside before you get lucky.
Hunters we spoke with were happy to see temperatures drop overnight because they say it gets the deer moving, increasing their chances of getting one.
"Yeah, it seems the deer move more when it's colder versus when it's warmer," said hunter Stephen Jessel. "That's why I am not complaining because they will be moving."
Some hunters said they wouldn't go home until they get a buck. Others felt differently, but they all agreed that there's nothing like being out in the woods.
"I guess you can get out of this world for a minute and just relax," Rodgers said.
"That's the serenity of a long work week," Patterson said.
"Just the drive, the feeling, shoot your deer and something to track them, I don't know. It's just in my blood, I guess," said hunter Austin Mangers.
Hunters can email their photos to email@example.com or by using our ReportIt! Feature by CLICKING HERE. We'll show some photos in our newscasts.


text: Copying and distributing are prohibited without the permission of the publisher. If you wish to distribute this content please contact Christopher Hunt (firstname.lastname@example.com) for a quote. Subscribe today: Order Euromoney by contacting us on +44 (0)20 7779 8999 (UK) or +1 212 224 3570. Rwanda reform drive on track as international debut bond takes off By: Matthew Turner Published on: Friday, April 26, 2013 Issuance reflects the scramble for African sovereign bonds and country's growing economic allure. Print Order Share Share on facebook Share on twitter Share on linkedin Share on googleplus Rwanda has become the latest sub-Saharan African (SSA) country to issue a debut international bond, underscoring its post-conflict rehabilitation in the eyes of investors and pent-up demand for the dwindling pool of hard-currency emerging market sovereign debt. The $400 million bond was issued with a yield of 6.875% with a 10-year maturity, to principally refinance infrastructure projects. Investor appetite for African sovereign bonds is reflected in Euromoneys Country Risk data, which shows an improvement in Rwandas access to capital markets (ATCM) indicator, up 0.5 points in the last quarter. ECRs ATCM indicator measures the ease to which a corporate or sovereign can access market finance. Rwanda central bank governor John Rwangombwa, in an interview with Euromoney Country Risk, explains why the sovereign shunned index buyers, and sheds light on economic reform efforts. Why did you decide not to issue a benchmark $500 million bond? We came to the market for one specific reason: to refinance infrastructure projects, so we didnt come to the market for the sake of borrowing for the budget deficit. We wanted really to finance various economic and structural projects, and the cost of these projects added up to $400 million. And when we reviewed the pricing, we thought the possible additional deficit point for not hitting the $500 million mark would be cheaper than taking just $100 million, which we didnt have a specific project finance. So it was very much a decision borne out of financing specific projects. How will this bond issue affect the countrys debt sustainability position? Out of the $400 million we are borrowing, it is only $200 million which is new debt and the other $200 million is financing. In terms of debt numbers, government debt is 24% of GDP. Rwanda central bank governor John Rwangombwa The IMF has endorsed our management style of the economy. We have ambitious programmes to develop our economy and we continue to implement reforms on an ongoing basis. Thats why we wanted to raise $400 million, as this figure was more applicable to our economic development and to financing specific projects. Will Rwandan economic policy be influenced by international market discipline? The debut bond issuance does bring another angle in terms of working with international investors in our economic affairs but I dont think it is something that will bring additional pressure that wasnt there before. The fact that we were able to sell our story shows that we are strong managers of the economy and committed to implementing new programmes. The projects will have a positive impact on the growth of our economy in general, but more so on increasing earning and living standards. Do you have plans for monetary reforms? We have financial sector reform plans that were approved by parliament before this bond issuance, so we have ongoing reforms. We have a five-year economic-development and poverty-reduction strategy, five different reforms in different sectors, and the financial sector and monetary system are integral to this plan. So there are no new specific reforms that we are doing in relation to this bond issuance. We maintain are reforms as planned, not just because we have come to the market. Are you satisfied with Rwandas B credit rating? Im not satisfied, because Id be happy to be an AAA country. But maybe thats where we are. We agree with the rating agencies about our weaknesses, which we are trying to address in the different measures we are taking. There were two core weaknesses affecting our credit rating. Firstly, a narrow export base, which was a challenge to us, so we have a new export promotion strategy, which was approved in 2011 and has seen our exports grow by 20% and we aim to expand on that. The other area highlighted was our high dependency on foreign aid, which was around 40% of GDP. But it is part of our government plan to reduce our dependency on government aid. So, overall Im not satisfied that we are a B country, but it is good that they show us where we are and to highlight our weaknesses, which we can improve on. Which corporates or state-backed organizations could issue in the international markets using the sovereign bond as a benchmark? We dont have any state-backed entities as we have privatized almost everything, but rather we hope private companies will take advantage of this. But it could be used by Rwandas national airline and Rwandan energy and utility agency. How does Rwandas bond issuance compare with other SSA sovereigns? Zambia had better terms than us because they are rated higher and their bond was related to their strong mineral base. So Id say we compare favourably in terms of the composition of our economy and financial sector in the region. To receive similar stories, sign up for Regions and emerging markets emails. Add Your Comment All fields are compulsory All comments are subject to editorial review as we are subject to the same regulations adhered to in publishing our own content. For this reason, your comment may not be live immediately, or may not be published. Your name: Your email: (will not appear) Publish your name? Comments: I have read and agree to the Terms and Conditions


text:
Is There a Dog in This Book?
Written by Viviane Schwarz
Illustrated by Viviane Schwarz
Lovereading4kids Price £9.59
£11.99 saving £2.40 (20%)
when in stock
As soon as this book is back in stock we will send you an email.Compare book price We'd love you to buy your books from Lovereading - especially if we helped you to choose them! But as our mantra is guidance, and that includes guidance on price, please do use our 'Compare Book Price' service to discover if the best value deal is from Lovereading or another specialist bookstore.
Please login (or signup) to download the extract
You will need to Sign Up or Login before you can download a book extract.
To Sign Up all you need to do is give us your email address and a password and let us know the types of books you like. Thank you.
The Lovereading4Kids comment
With an irresistible story that directly involves the reader, this book's clever design and bright, gestural illustrations make for a LOT of fun for cat (and dog) lovers! It is a completely interactive book brimming over with all kinds of fantastic flaps and fabulous flips that reveal lots of little surprises and jokes for children to enjoy and even for parents to share with their toddlers too!
Is There a Dog in This Book? by Viviane Schwarz
In this third title in the extraordinarily inventive, brilliantly interactive Cats series from award-winning illustrator Viviane Schwarz - our three favourite picture book cats Tiny, Moonpie and Andre make a new furry friend...Something smells funny...what can it be? Wait, hang on a second, is it a DOG? Is there a dog in this book?! Oh no! Tiny, Moonpie and Andre are SCARED of dogs! Dogs are snappy and yappy, smelly and noisy and...they HATE cats. So, they all try and hide from him - behind the couch, under the piano, in the wardrobe - but when the little doggy finally finds them, well, they simply can't get enough of him! He is really very soft. And they want YOU to love him, too - go on, just give him a little cuddle.
About the Author
Other Formats
Book Info
32 pages
Interest Age: From 3
Viviane Schwarz
More books by Viviane Schwarz
Author's Website
Walker Books Ltd
Publication date
1st September 2014
I love Lovereading because I often never know what to read and this helps you find a good book. I have recommended this to many people.Shannon-Louise Masters – age 15
We love Lovereading4kids because they put books in front of us we wouldn't otherwise have read. They make us more adventurous readers!Emily Jacques
LoveReading4Kids is a modern and creative way of emphasising the value and importance of books in this digital age #booksforlifeAmrit Bunet – Teen
We love Lovereading4kids because they put books in front of us we wouldn't otherwise have read. They make us more adventurous readers!Emily Jacques
A great way to introduce kids to great books, authors & genres. Parents can find age-appropriate books to share with their children.Judi Davies – Aberdare Girls Sch
At @HHSHaringey we love @lovereading4kids because our pupils can practice reviewing & get free upcoming books before anyone else!Helen Swinyard – Heartlands High
I think Lovereading4kids is an amazing company because of the friendly staff and the fabulous chance to read great books before publication.Adam Graham
It is fantastic, you get to read lots of books and you always find something new and amazing in them.Erica Motoc, age 7


text:
Woman arrested in California for 2007 Topeka murder
A woman wanted by the Shawnee County Sheriff's office for second degree murder was arrested on Wednesday in California.
A woman wanted by the Shawnee County Sheriff's office for second degree murder was arrested on Wednesday in California.
Two arrests have been made in connection with an armed robbery case that led to the lockdown of schools in Wamego.
Some roads in Northeast Kansas are still closed due to flooding.
Authorities say a 7-year-old girl has died after being pulled from a river in southeastern Kansas.
A man charged in the slaying of a Wichita couple during a robbery has pleaded guilty.
The latest claimant in what could grow into a long line of people asserting that they're entitled to a piece of the musician's fortune.
Internet giant Google said Wednesday it will ban all ads from payday lenders, calling the industry "deceptive" and "harmful."
Kansas has delayed cutting off Medicaid funding for Planned Parenthood a second time and postponed its action against the abortion provider …
Three teens from a Kansas military school are dead and another is injured after their pickup crashed in the foothills outside Denver.
For decades, pregnant women and women who may become pregnant have been advised to take folic acid to help prevent certain birth defects.
Tony Kirk's son, Kalen, was hit by a crossing guard supervisor's vehicle. It happened in Lakeland, a city in Central Florida.
One New Jersey teacher is finding a way to make test-taking personal. And, her messages of support are spreading across the globe.
A fire that caused an explosion at a Texas fertilizer plant that killed 15 people in 2013 was a criminal act.
Funeral services have been set for a form Seaman School District Principal who was found deceased in a downtown Topeka warehouse Monday morn…
The intentional killing of a police officer carries a possible death sentence in Kansas.
A company in Topeka can say they're one of the safest companies in the city.
NASCAR has suspended Kyle Busch's crew chief and tire changer for one race for violating the lug nut policy during the team's win at Kansas …
A man described by his family as mentally disturbed went on a stabbing rampage hours after leaving a hospital, killing two people and assaul…


text:
(Photo: Volunteers nominated for their exceptional contribution during 2016- Congratulations Nora, Chris, Yvonne (overall winner), Maria and Vera )
Become involved with your local community health service
VOLUNTEER with US!
Volunteers assist us in establishing stronger links with our client communities. Volunteers are highly regarded for their generous time, skills, commitment and dedication to help us provide quality services as we strive for our vision of a healthy and inclusive community.
Why volunteer at ISCH?
- be part of a dedicated and innovative workforce
- build self confidence
- meet new people
- give back to the community by helping others
- learn new skills
- stay active
- have FUN!
Read the latest edition of our volunteer newsletter Volunteer News – 4th Edition 2016
Volunteer Opportunities Available:
Our volunteers are involved in a variety of different roles, ranging from administration, client support, dietetics, Indigenous programs, exercise groups, mentoring and graphic design to name just a few.
We are NOT currently looking for more volunteers in any areas at the moment (updated February 2017):
Volunteer Requirements:
- Undergo a National Volunteer Police Check (paid by ISCH)
- Complete a Volunteer Working With Children Check (if required by your role)
- Complete volunteer induction training
- Attend relevant training and information sessions (dependent on your role)
- Sign and agree to our Code of Conduct and Confidentiality terms and conditions
How To Become A Volunteer With Us:
To join our dedicated team of volunteers, you can get started by completing the following:
- Volunteer Registration
- If you are unable to complete, please call 9525 1300.
Thank you for your interest in volunteering with us. We hope to see you soon!
- Community Directory Support socially inclusive cafes & organisations in our local area, using the ISCH 'Welcome' directory
- Check out ISCH on LinkedIn Visit the ISCH LinkedIn page for updates on current vacancies and information about working with us!
- Trauma and Homelessness Initiative ISCH has been involved in some very important work around trauma and homelessness. Read about it here.
- SHIP Social Health & Inclusion Port (SHIP) Residents and organisations working together to create a healthy and inclusive Port Melbourne community.
- Case Management Learn more about how our skilled team of case managers can assist you.
- Housing and Homeless Supporting people who are homeless or at risk of homelessness is a focus of our services.
- Group Work View our Group Work page for a group program that might be right for you.


text:
Already a paid subscriber but not registered for online access yet? For instructions on how to get premium web access, click here.
Home / News / Healthcare / Davis Vision moving HQ to Texas
Get our free LIBN e-alerts & breaking news notifications!
Vision insurance company Davis Vision is relocating its headquarters from Plainview to San Antonio, Texas, a move expected to affect about 100 administrative employees on Long Island. The company, which is relocating to be closer to its parent company's base, said the move won't affect the more than 1,300 other Davis Vision and Empire Vision retail ...
Tagged with: Long Island
3 comments
And the exodus continues. Manufacturing division will be next. Is there any wonder?
Of course manufacturing will be next as will pretty much the rest of the company save retail. This is another one of those "Six Sigma" beancouter moves that ignores the tradition and the quality people that made this company profitable. This is a trade of lower short-term costs at the expense of long term profit and growth.
Bottom line is I will be one of over a hundred Davis Vision associates in Long Island out of a job come September 2011 thanks to HVHC.


text:
5 Hair Care Myths Debunked
When it comes to hair care, there is a lot of information out there, some reasonable and some that seems downright crazy.
Save for trying out every tidbit of hair wisdom that's out there on your locks, how's a girl to differentiate fact from fiction in her quest to get the gorgeous strands she craves?
Misinformation can be just as stubborn as frizz or those pesky flyaways – it's difficult to manage, impossible to reason with and it just keeps coming back, says longtime hair-care advocate and health scientist Audrey Davis-Sivasothy, author of Hair Care Rehab. Read more …
5 Signs You Belong Together
We've all heard the old saying that there's someone out there for everyone. Here are a few signs you can look for to figure out just how well you and your partner mesh together.
3 Reasons to Stay Single
There are plenty of benefits to being in a relationship, but there are just as many reasons for staying single.
9 Sexual Communication Tips
Talking about sex can be stressful, Here are nine helpful tips to make it easier.
Top 5 Gifts for Couples
Are you looking for something you can share with your partner? Check out these great recommendations to find something that you'll both love.
Top 5 Gifts for Him
Not sure what to get your man? Check out some of the top gift ideas from the guys at Adam & Eve.
Indulge in a little "me time"
Adam & Eve Adult Toys. Always Discreet, Free Shipping and Bonus Gifts.


text:
Drawing and sketching anime steampunk mecha girl (woman) Part 2 or 4 for manga, cartoon and comics. Learn how to draw woman body (girl or female figure): Step by Step video tutorial. Drawing girl body (torso and pelvis)
Learn from basic sketching to finish drawing quick concept for comics cartoon illustration, manga, anime.
Here is a final sketch:
-Fine tuning line drawn for female form and anime face.
- Final drawing over rough sketch lines.
If you miss part 1go here:
-Drawing Mecha Steampunk Anime Girl part 1
Here is the Video part 2, enjoy!
Drawing Software & Tools I used:
-E frontier Manga Studio EX Professional
-Genius MousePen 8X6 Graphic Tablet for Home and Office
*It could also be done with Graphite or Pencil, gauche and Paper.
New tutorials:
-Drawing Monkey King character Part 1 (drawing and sketching ideas)
-Drawing Monkey King character Part 2 (Establishing light and shadow or value)
-Drawing Monkey King character Part 3 (Assign Color and composition)
Recommended tutorials:
-Drawing woman character: "Assassin" action pose for comics, manga , and illustration
-Drawing Girl: Dragon Slayer for manga, comic, and illustration
-Drawing and Painting Futuristic girl superhero for comics
-How to draw manga anime girl with yoyo.
-How to draw basic Mecha robot
More Basic People drawing:
-Learn how to draw Female face front view
-Learn how to draw Female face 3/4 view
-Drawing Face Profile (Sideview) Female
-How to draw & sketch hands: female, girl, woman.
-Basic Drawing Lessons
Special thanks to these sites for their kind words and link to our site:
- AnotherHere (Cool Blog)
- Pixelame.net (Cool Social Bookmark Blog)
-Blender Artist
-TokyoPop.com (Big Thanks for featuring on of my VIDEO!!!)
-TokyoPop Japan
-AmethystBlog (Thanks for the Great Review!)
Tuesday, November 27, 2007
Learn how to draw step by step: anime mecha steampunk girl for manga comics part 2
Drawing and sketching anime steampunk mecha girl (woman) Part 2 or 4 for manga, cartoon and comics. Learn how to draw woman body (girl or female figure): Step by Step video tutorial. Drawing girl body (torso and pelvis)
Popular Posts
Concept art waterfall bridge environmental design. How to use PS custom water brushes in your concept design. I post a PS brushes link y...
How to draw hand video tutorial learn step by step basic. Now back to the basic a little bit. I have so many emails, youtube comment, ...
Learn how to draw woman, female, girl body: Figure Step by Step sketching and rendering online lesson.Tips on drawing female body . Learn how to draw woman, female, girl body: Figure Step by Step sketching and rendering online lesson. Dra...
7 most useful digital painting tips for artists. These are the most requested or most asked questions that I get for the past five to six ...
How to draw lips (mouth): girl, women, lady. Realistic lips tutorial step by step. There is a request so here is goes. I don'...
How to draw a lion . Drawing a lion head study. It's been a while since I've draw or sketch any creature so I decided to sketch...
How to draw fist hand 5 different ways step by step and video tutorial. In this tutorial, I will show you the easiest way possible t...
How to draw comics page layout . Manga process tutorial step #1 thumbnails page layout story telling. How to draw Manga step by step. ...
How to draw a nose step by step front view facial video tutorial. Another facial structure sketch and drawing study. Now I am kind...
How to draw hand holding gun . This tutorial have two different parts. The first part is drawing a hand holding a pistol from forehan...
art lesson online
Recommended Tutorials and Books
Join our Affiliate Program / Terms of use / Private Policy /About us / Contact Us©2007-2013 All Rights Reserved. All the images here and idrawgirls.com are trademarks used by CreativMobiLLC & Xia Taptara.


text:
Hello all !
You're new in town or an old inhabitant of Clint City, introduce yourself here.
Who are you ? When did you arrive in town ? What is the name of your beloved pet ? Do you prefer chocolate ice cream or sauerkraut hot dogs (with chocolate too if you want) ?
This thread is not made to recruit people in your guild, just to know each other better
HI u suck i am the best
Hey I am Laguna. I just joined today.
Hullo... im Bad Santa and despite popular belief im actually from the capital of ireland. been playing a couple o weeks now and starting to get the hang of it
I'm that boy replied from the number 86, I'm Peter aka RpgMastEra, Now 12 yrs old, Manhattan, New York, US of A Catholic, love all that stuff but except for wrestling, like the young mafia, favorite thing: girls, i'm trying to look for a guild that accepts my speech.
Yo!My name Victor and from two days ago I started playing this game.I gotten pretty good with some luck and skill but mostly luck.As you can see from my avatar I like Afro Samuri but anime ever.hahaha.But my favorite group is the Bangers and I'm tryin to create a deck based on a lil of that.
So add me as friend or add me to a guild I'm here to be around.
Hi, i'm new.


text: WASHINGTON — A group of House Republicans called Wednesday for a congressional investigation into the improper handling of classified documents by President Clinton's national security adviser, Sandy Berger. Berger admitted last year that he deliberately took classified documents out of the National Archives in 2003 and destroyed some of them at his office. He pleaded guilty in federal court to one charge of unauthorized removal and retention of classified material and was fined $50,000. STORY LINK And who says that the Republican won't hash out old news to try to win! I mean, he pleaded guilty in 2003! 3 Years ago! It's it funny how it just becomes news again... Refresh people's memories weeks befor the election. Jamie Sad! Truely sad! All Politicans are corrupt!


text:
Is May Ganton Beauty Salon incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
Are these details correct?
Hotfrog Philippines provides information regarding May Ganton Beauty Salon in Mabalacat Pampanga. May Ganton Beauty Salon is located at 167-B, MacArthur Highway, Mabalacat Pampanga 2010 and provides Personal Care services. Contact them on (045) 892 4420.
Is May Ganton Beauty Salon in 167-B, MacArthur Highway, Mabalacat Pampanga 2010 your business? Claim your listing and attract more leads by adding more content, photos and other business details.
We have more Personal Care services in Mabalacat Pampanga available on Hotfrog Philippines. You can update your search for Mabalacat Personal Care by location, keyword or service options.
Small Business Hub
Sharing tips, ideas and advice to help grow your small business.
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub


text:
The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection
Cheng H.-Q.,CAS Institute of Microbiology |
Cheng H.-Q.,State Key Laboratory of Plant Genomics |
Cheng H.-Q.,University of Chinese Academy of Sciences |
Han L.-B.,CAS Institute of Microbiology |
And 15 more authors.
Journal of Experimental Botany | Year: 2016
Accumulating evidence indicates that plant MYB transcription factors participate in defense against pathogen attack, but their regulatory targets and related signaling processes remain largely unknown. Here, we identified a defense-related MYB gene (GhMYB108) from upland cotton (Gossypium hirsutum) and characterized its functional mechanism. Expression of GhMYB108 in cotton plants was induced by Verticillium dahliae infection and responded to the application of defense signaling molecules, including salicylic acid, jasmonic acid, and ethylene. Knockdown of GhMYB108 expression led to increased susceptibility of cotton plants to V. dahliae, while ecotopic overexpression of GhMYB108 in Arabidopsis thaliana conferred enhanced tolerance to the pathogen. Further analysis demonstrated that GhMYB108 interacted with the calmodulin-like protein GhCML11, and the two proteins form a positive feedback loop to enhance the transcription of GhCML11 in a calcium-dependent manner. Verticillium dahliae infection stimulated Ca2+ influx into the cytosol in cotton root cells, but this response was disrupted in both GhCML11-silenced plants and GhMYB108-silenced plants in which expression of several calcium signaling-related genes was down-regulated. Taken together, these results indicate that GhMYB108 acts as a positive regulator in defense against V. dahliae infection by interacting with GhCML11. Furthermore, the data also revealed the important roles and synergetic regulation of MYB transcription factor, Ca2+, and calmodulin in plant immune responses. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. Source
Wang F.-X.,CAS Institute of Microbiology |
Wang F.-X.,State Key Laboratory of Plant Genomics |
Ma Y.-P.,CAS Institute of Microbiology |
Ma Y.-P.,State Key Laboratory of Plant Genomics |
And 11 more authors.
Proteomics | Year: 2011
Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source
Proteomic identification of differentially expressed proteins in the Ligon lintless mutant of upland cotton (Gossypium hirsutum L.)
Zhao P.-M.,CAS Institute of Microbiology |
Zhao P.-M.,State Key Laboratory of Plant Genomics |
Zhao P.-M.,National Center for Plant Gene Research |
Wang L.-L.,CAS Institute of Microbiology |
And 19 more authors.
Journal of Proteome Research | Year: 2010
Cotton fiber is an ideal model for studying plant cell elongation. To date, the underlying mechanisms controlling fiber elongation remain unclear due to their high complexity. In this study, a comparative proteomic analysis between a short-lint fiber mutant (Ligon lintless, Li 1) and its wild-type was performed to identify fiber elongation-related proteins. By 2-DE combined with local EST database-assisted MS/ MS analysis, 81 differentially expressed proteins assigned to different functional categories were identified from Li 1 fibers, of which 54 were down-regulated and 27 were up-regulated. Several novel aspects regarding cotton fiber elongation can be illustrated from our data. First, over half of the downregulated proteins were newly identified at the protein level, which is mainly involved in protein folding and stabilization, nucleocytoplasmic transport, signal transduction, and vesicular-mediated transport. Second, a number of cytoskeleton-related proteins showed a remarkable decrease in protein abundance in the Li 1 fibers. Accordingly, the architecture of actin cytoskeleton was severely deformed and the microtubule organization was moderately altered, accompanied with dramatic disruption of vesicle trafficking. Third, the expression of several proteins involved in unfolded protein response (UPR) was activated in Li 1 fibers, indicating that the deficiency of fiber cell elongation was related to ER stress. Collectively, these findings significantly advanced our understanding of the mechanisms associated with cotton fiber elongation. © 2010 American Chemical Society. Source
Cotton major latex protein 28 functions as a positive regulator of the ethylene responsive factor 6 in defense against verticillium dahliae
Yang C.-L.,CAS Institute of Microbiology |
Yang C.-L.,State Key Laboratory of Plant Genomics |
Yang C.-L.,University of Chinese Academy of Sciences |
Liang S.,CAS Institute of Microbiology |
And 18 more authors.
Molecular Plant | Year: 2015
In this study, we identified a defense-related major latex protein (MLP) from upland cotton (designated GhMLP28) and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jasmonic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing resulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 (GhERF6) and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the transcription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants. © 2015 The Author. Source
The thioredoxin GbNRX1 plays a crucial role in homeostasis of apoplastic reactive oxygen species in response to verticillium dahliae infection in cotton
Li Y.-B.,CAS Institute of Microbiology |
Li Y.-B.,State Key Laboratory of Plant Genomics |
Li Y.-B.,University of Chinese Academy of Sciences |
Han L.-B.,CAS Institute of Microbiology |
And 16 more authors.
Plant Physiology | Year: 2016
Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae. Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response. © 2016 American Society of Plant Biologists. All rights reserved. Source


text:
Gilbert and Sullivan's London
By (author) Andrew Goodman
Foreword by Mike Leigh
Format: Paperback
Normal Price: $35.00
Your Price: $31.50 AUD, inc. GST
Shipping: $7.95 per order
You Save: $3.50! (10% off normal price)
Plus...earn $1.58 in Boomerang Bucks
Availability: Available to Backorder, No Due Date for Supply
Gilbert and Sullivan's London by Andrew Goodman
Book DescriptionAndrew Goodman tells the story of the most astonishing partnership in the history of British musical entertainment, and unfolds it against the authentic background of the city and era in which they flourished. With much original research and many hitherto unpublished illustrations, Goodman makes an invaluable contribution to our knowledge, understanding and appreciation of Gilbert and Sullivan in this handsome volume, already a classic of its kind. He summons up a bygone era in a predominantly celebratory tone: recalling the lost grandeur of the city's great entertainment palaces, its atmospheric streetlife and nightlife, its splendour and its squalor. Mike Leigh, director of "Topsy Turvy", a film about Gilbert and Sullivan's collaboration on "The Mikado", provides a new foreword.
Buy Gilbert and Sullivan's London book by Andrew Goodman from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9780571200160
(245mm x 188mm x 13mm)
Pages: 192
Imprint: Faber & Faber
Publisher: Faber & Faber
Publish Date: 17-Apr-2000
Country of Publication: United Kingdom
Other Editions...
Books By Author Andrew Goodman
Effective Written Advocacy, Paperback (February 2012)
This book not only offers a practical and comprehensive guide to effective written advocacy, but provides worked examples drawn from real cases contributed from today's leading and highly successful advocates.
Preparing for Mediation, Paperback (November 2011)
Most disputants coming to mediation are unlikely to be familiar with the process, and wary of what may seem to be a strange procedure, particularly the idea that it is voluntary, non-adjudicative, non-binding until its conclusion, confidential, and driven entirely by the wishes of the parties. This book provides the answers.
Court Guide to the South Eastern and Western Circuits 2006/2007, Paperback (April 2006)» View all books by Andrew Goodman
Includes details of courts on both the South Eastern Circuit and Western Circuit. Designed to aid those who use the courts, this guide provides contact details for over 700 courts, including various Crown, County, Magistrates', and more. It contains information such as travel directions, court sitting times, facilities available, and hotels.
» Have you read this book? We'd like to know what you think about it - write a review about Gilbert and Sullivan's London book by Andrew Goodman and you'll earn 50c in Boomerang Bucks loyalty dollars (you must be a member - it's free to sign up!)
Facebook Page | Twitter | Google+ | Bulletin e-Newsletter | Blog | RSS
Become a Member | Book Lists | Recently Released | Coming Soon | Fast Delivery Books
Bestselling Books: Our Current Bestsellers | Australia's Hottest 1000 Books | Bestselling Fiction | Bestselling Crime Mysteries and Thrillers | Bestselling Non Fiction Books | Bestselling Sport Books | Bestselling Gardening and Handicrafts Books | Bestselling Biographies | Bestselling Food and Drink | Bestselling History | Bestselling Travel Books | Bestselling School Textbooks & Study Guides | Bestselling Children's General Non-Fiction | Bestselling Young Adult Fiction | Bestselling Children's Fiction | Bestselling Picture Books | Top 100 US Bestsellers
For: Schools | Organisations | Libraries | Publishers | Authors | Book Clubs | Bloggers | Affiliates
About Us | Contact Us | Help Desk | Security | Privacy | Terms of Use | Copyright
Phone: 1300 36 33 32 (9am-5pm Mon-Fri AEST) - International: +61 2 9960 7998 - Online Form
Address: Boomerang Books, 878 Military Road, Mosman Junction, NSW, 2088
© 2003-2017. All Rights Reserved. Eclipse Commerce Pty Ltd - ACN: 122 110 687 - ABN: 49 122 110 687
For every $20 you spend on books, you will receive $1 in Boomerang Bucks loyalty dollars. You can use your Boomerang Bucks as a credit towards a future purchase from Boomerang Books. Note that you must be a Member (free to sign up) and that conditions do apply.


text:
I agree Our site saves small pieces of text information (cookies) on your device in order to deliver better content and for statistical purposes. You can disable the usage of cookies by changing the settings of your browser. By browsing our website without changing the browser settings you grant us permission to store that information on your device.
|Price ($)
Tributyl phosphite may be employed as model lubricant additive and its surface chemistry on Fe3O4 in ultrahigh vacuum is investigated. It is used as lubricants, preservatives, heat stabilizers of intensifier.
Feng Gao; Octavio Furlong; Peter V Kotvis; Wilfred T Tysoe. Reaction of tributyl phosphite with oxidized iron: surface and tribological chemistry. Langmuir. 2004, 20, (18), 7557-7568.
A M Saboori; D M Lang; D S Newcombe. Structural requirements for the inhibition of human monocyte carboxylesterase by organophosphorus compounds. Chemico-Biological Interactions. 1991, 80, (3), 327-338.
Hazard Statements: H312-H315-H319-H227-H303
Harmful in contact with skin. Causes skin irritation. Causes serious eye irritation. Combustible liquid. May be harmful if swallowed.
Precautionary Statements: P210-P280-P305+P351+P338-P362-P312-P363-P321-P322-P403+P235-P501a
Keep away from heat/sparks/open flames/hot surfaces. - No smoking. Wear protective gloves/protective clothing/eye protection/face protection. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Take off contaminated clothing and wash before reuse. Call a POISON CENTER or doctor/physician if you feel unwell. Wash contaminated clothing before reuse. Specific treatment (see label). Specific measures (see label). Store in a well-ventilated place. Keep cool. Dispose of contents/container in accordance with local/regional/national/international regulations.


text: Seller: tampa_art (2,422) 100%, Location: Tampa, Florida, Ships to: Worldwide, Item: 152333226788 Vintage. Pre-owned. Very good condition. Has one chip along the top (see photographs) Vintage Large Davidson Clear Glass Flower Dome / Support / Block / Holder / FrogMarked on the underside: Made in England. Patent 7830 / 1910Oval. About 8 inches x 5 inches x about 4 1/2 inches high. Collectible gardening accessory.WEIGHT: About 3 pounds, 3 ounces (on a postal scale)CONDITION: Pre-owned. Mint to Very good condition. One chip along the top---see photographs. Small area of impact fracture along the bottom---does not impact use or function.Buyer pays $9.95 for USPS Priority shipping to all addresses in the USA. Delivery confirmation included.International shipping is extra and based on weight and location.Seller accepts PayPal.Please see my other active auctions. Discount shipping is available for all purchases sent to the same address.ALL SALES FINAL. Please email me with any questions. Condition: Pre-owned. Very good condition. One chip. See condition notes., Color: Clear, Material: Glass, Year: 1910, Country: England, Maker: Davidson