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Conceived and designed the experiments: DVA IYZ NAG. Performed the experiments: DVA IYZ. Analyzed the data: DVA IYZ NAG. Wrote the paper: JMV NAG.
E-cadherin–mediated cell–cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell–cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin–based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell–cell interactions, preventing contact paralysis after the establishment of cell–cell contact and promoting dynamic cell–cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells.
Analyses of cell-cell interactions and of the establishment of cell-cell adhesion are important directions in the studies of normal morphogenesis and its pathological alterations during tumor progression. Long ago, Abercrombie coined the term 'contact inhibition of locomotion' [1] for a phenomenon that has recently been described for neural crest cell migration in vivo [2]. The nature of this phenomenon and of changes of cell-cell interactions of neoplastic cells has still not been elucidated. Classical cadherins are transmembrane proteins that mediate cell-cell adhesion through Ca+2-dependent homophilic interactions of their ectodomains. The intracellular domain of the cadherin molecule interacts with proteins of cytoplasmic plaque (catenins, vinculun) that link it to actin filaments to maintain the stability of adherens junctions (AJs). The stable E-cadherin-based AJs play a pivotal role in the integrity of the epithelium and the maintenance of tissue homeostasis [3]. Cell-cell adhesion is often modified during cancer progression [4]. Epithelial-mesenchymal transition (EMT), which includes the downregulation of E-cadherin expression, the disruption of cell-cell contacts, and the acquisition of an elongated fibroblast-like phenotype by epithelial cells, has been observed in many carcinoma cell lines. It has been thought for a long time that a decrease in the E-cadherin level on the cell surface in the course of EMT is a key step of the progression from adenoma to carcinoma [5]. Many tumors, however, continue to express E-cadherin [6], and thus there should be other mechanisms to regulate the E-cadherin adhesive function in carcinomas. The early stages of the morphological transformation of epithelial cells, including rearrangements of the actin cytoskeleton and cell-cell interactions, have not yet been characterized.
It has been shown that Rho family small GTPases, which regulate cytoskeletal dynamics [7], are involved in cadherin-dependent adhesion. In epithelial cells, inhibition of endogenous Rho or Rac decreased accumulation of E-cadherin at cell-cell contacts [8], [9]. Rac1, which regulates Arp2/3-mediated actin polymerization, is associated with the newly formed cell-cell contacts [10]. Early AJs incorporate exogenous G-actin and depend on actin polymerization [11], [12]. The Arp2/3 regulators N-WASP, WAVE2, and cortactin are involved in the assembly of AJs [12]–[15]. Rho activity is required for the formation of actin stress fibers and focal contacts [7]. Rho-kinase (ROCK) and the formin protein mDia1 are the main effectors of Rho [16]–[18]. Active Rho recruites ROCK, which stimulates acto-myosin contractility by phosphorylation of the myosin II light chain and other targets [19]. Data on the role of myosin II-mediated contractility in the establishment of cadherin-mediated adhesion are controversial. Shewan et al. [20] suggested that myosin II is essential for the accumulation of E-cadherin at cell-cell junctions. In other studies, the ROCK inhibitor Y-27632 and the myosin II ATPase inhibitor blebbistatin had no effect on the assembly of AJs in epithelial cells [12], [21]. Formin-mediated actin polymerization could also be important for the establishment of AJs. Formin1 is essential for cadherin-mediated adhesion in keratinocytes [22]. It has also been shown that mDia1 participates in the formation and maintenance of AJs in MCF7 epithelial cells [23].
In this work, we studied the lines of transformed epithelial cells that retained expression of E-cadherin, displayed slightly changed morphology, and formed monolayers in dense culture. In transformed epithelial cells, we found, however, a disappearance of the marginal actin bundle and dramatic rearrangement of E-cadherin-based AJs into radial AJs that are differently regulated by Rho family small GTPases than are tangential AJs in nontransformed epithelial cells. We also described the remodeling of radial AJs and the alterations in protrusive activity that led to reduced cell-cell adhesion and to acquisition of motile behavior and migratory capacity by transformed epithelial cells.
In this study, we compared the morphology, the actin cytoskeleton, and AJs of nontransformed IAR-2 epithelial cells derived from rat liver with those of the transformed IAR descendants IAR-6-1 and IAR1170. In dense culture, nontransformed IAR-2 epithelial cells formed a monolayer (Figure 1A). Individual IAR-2 cells were discoid-shaped. They had a marginal actin bundle, which is typical of epithelial cells and internal straight actin bundles (Figure 1B). Immunofluorescence microscopy of IAR-2 cells stained with antibodies against E-cadherin showed that AJs were aligned as a continuous belt along the cell-cell boundaries and colocalized with the circumferential actin belt (Figure 1C and 1D). We designated AJs of nontransformed epithelial cells 'tangential AJs'.
Most descriptions of transformed epithelial cells in vitro focus on cell lines that underwent EMT. We, however, decided to study transformed epithelial cell lines that had a slightly changed morphology. We chose an IAR epithelial line transformed in vitro with dimethylnitrosamine (IAR-6-1). As previously reported [35], the IAR-6-1 line was tumorigenic in syngenic rats. In comparison with IAR-2 cells, the morphology of IAR-6-1 cells was minimally changed. Single IAR-6-1 cells were polygonal or discoid. In confluent culture, IAR-6-1 cells formed a monolayer similar to the IAR-2 monolayer (Figure 1E and 1F). Western blot analysis showed that transformed IAR-6-1 epithelial cells retained expression of epithelial E-cadherin and did not express mesenchymal N-cadherin (Figure S1). Differences between nontransformed IAR-2 cells and transformed IAR-6-1 cells became evident when they were fluorescently stained for the actin cytoskeleton and E-cadherin. In contrast with nontransformed IAR-2 cells, IAR-6-1 cells had only randomly oriented actin bundles in the cytoplasm; the marginal actin bundles had disappeared (Figure 1F). We also revealed the dramatic rearrangement of E-cadherin-based AJs in IAR-6-1 cells that were organized into strands arranged perpendicularly or at different angles to the cell edge (Figure 1G). We designated AJs of transformed epithelial cells 'radial AJs'. Double fluorescence staining showed that AJs in transformed IAR-6-1 cells were colocalized with short straight actin bundles (Figure 1G and 1H). In dense cultures, some AJs were oriented along the cell-cell borders.
We also established 14 clones of IAR-2 cells transformed with Ras and compared their morphology. Cells of nine Ras-transformed clones (IAR1162 clones) underwent EMT: they lost the epithelial phenotype and acquired fibroblastic-like morphology. As Western blotting showed, IAR1162 cells did not expressed epithelial E-cadherin and did express mesenchymal N-cadherin (data not shown). We investigated, however, the other five clones of Ras-transformed IAR-2 cells (IAR1170-D6, -D11, -F9, -H5, and -D12), which in comparison with nontransformed IAR-2 cells had slightly changed morphology. Ras-transformed IAR1170 cell lines were tumorigenic in nude mice (Figure S2). All five clones had similar morphology. The shapes of IAR1170 cells were near-discoid or polygonal. In confluent culture, IAR-1170 cells formed monolayers. As an example, we give images of IAR1170-H5 clone (Figure 1I and 1J). Western blot analysis showed that cells of IAR1170 clones retained E-cadherin and also expressed N-cadherin (Figure S1). A characteristic feature of the actin cytoskeleton in IAR1170 cells was the disappearance of the marginal actin bundle (Figure1J). In IAR1170 cells, we also revealed the reorganization of E-cadherin-based AJs. The continuous adhesion belt that was typical of IAR-2 cells was destroyed. AJs stained for E-cadherin were seen as punctate clusters at the cell-cell boundaries. The circumferential actin belts were disorganized (Figure 1K and 1L).
Thus, we described the early stages of the morphological transformation of epithelial cells, when cells retained expression of E-cadherin, had discoid or polygonal morphology, and formed monolayers. Neoplastic transformation, however, led to the disappearance of the marginal actin bundle and to the dramatic rearrangement of E-cadherin-based AJs.
As neoplastic transformation leads to disappearance of the marginal actin bundle and to rearrangements of AJs in epithelial cells, we decided to test whether these alterations contribute to cell-cell interactions of transformed IAR-6-1 and IAR1170 cells. To compare cell-cell interactions in cultures of nontransformed and transformed cells, sparse cultures of IAR-2, IAR-6-1, and IAR1170 cells were observed with time-lapse videomicroscopy. Nontransformed IAR-2 cells in sparse cultures formed islands. Time-lapse imaging showed that cell-cell contacts in these islands were stable (Figure 2A and Video S1) and were disrupted only during mitosis. Contacts between neighboring islands or single IAR-2 cells led to the establishment of strong cell-cell adhesion and development of a continuous monolayer for 2–3 days.
In cultures of transformed IAR-6-1 and IAR1170 cells, we revealed significant alterations in cell-cell interactions. In contrast with nontransformed IAR-2 cells, cell-cell contacts in cultures of transformed IAR-6-1 and IAR1170 cells were unstable and often broke (Figure 2B and 2C, Video S2, and Video S3). Transformed cells in contact extended lamellipodia at free edges. During 6–7 h of observation, the same cell could establish and break contacts with different neighboring cells and could move in different directions. This behavior of transformed epithelial cells may promote their migratory activity.
To explore the effects of changed motile behavior of transformed IAR-6-1 and IAR1170 cells on their migration, the ability of cells to migrate through polycarbonate membrane inserts with 8-µm pores in Bio-Coat migration chambers was examined. Migration assay showed that the number of migrating IAR-6-1 and IAR1170 cells was higher than that among nontransformed IAR-2 cells (Figure 3). Thus, disappearance of the marginal actin bundle and rearrangement of E-cadherin-based AJs is accompanied by reduction in cell-cell adhesion and promotion of migratory activity of transformed cells.
For detailed analysis of cell behavior during formation of contact with neighboring cells, nontransformed IAR-2 cells and transformed descendants were imaged in a narrow wound (~100 µm) in a monolayer. As IAR1170 expressed both E- and N-cadherin and there are findings that N-cadherin promotes motility when expressed by epithelial cells [24], for detailed studies we focused on IAR-6-1 cells, which expressed E-cadherin and did not express mesenchymal N-cadherin. We made narrow wounds by scraping cell monolayers with a sterile needle. After 3–4 h of incubation, cells from adjacent monolayers began to form new cell-cell contacts in the area of colliding lamellipodia. In the case of nontransformed IAR-2 cells, there was lateral expansion of initial stable contact between opposing cells (Figure 4A and Video S4). During the first 5–10 min after the establishment of a stable contact, cells formed lamellipodia both in the zone of contact and at the free edges. Within the next 15–20 min, dramatic cessation of protrusive activity along the entire contact (contact paralysis) developed. It began simultaneously along the entire length of the contact. The cells formed small lamellipodia only at the edges of the contact. Contact paralysis did not extend to the free edges of contacting cells. We observed, however, a decrease of protrusive activity at the free edges of contacting cells. We analyzed protrusive activity using kymographs (Figure 4B). Quantitative analysis of kymographs showed that both phases of the protrusion-retraction cycle, especially protrusions, were affected during contact paralysis. The rate of lamellipodial protrusion at the site of the contact was strongly diminished by 15–20 min after the establishment of the contact. The rate of lamellipodial retraction at the site of the contact was also reduced. Analysis of kymographs also showed statistically significant decrease of the mean rate of lamellipodial protrusion at the free edges of contacting cells. (Figure S3).
Neoplastic transformation modified the dynamics of cell-cell interactions in a narrow wound. After the establishment of the contact between two cells, transformed IAR-6-1 cells continued to form lamellipodia both at the site of the contact and at the free edges (Figure 4C and Video S5). The rates of lamellipodial protrusion and retraction did not change (Figure 4D and Figure S3). We did not see contact paralysis of protrusive activity at the site of the contact of IAR-6-1 cells (Figure 4D), and further analysis revealed significant overlapping in this zone (Figure 4E). While the mean overlapping area of contacting IAR-2 cells was 18.0±2.93 µm2 (mean±SEM, n=12), the mean overlapping area of contacting IAR-6-1 cells was 76.5±17.37 µm2 (n=14) at 15 min after cell-cell contact establishment.
The studies performed demonstrated the differences in cell behavior during the establishment of cell-cell contact by nontransformed and transformed cells. Cell-cell interactions of nontransformed epithelial cells result in the establishment of stable cell-cell contact followed by contact paralysis at the site of the contact and a decrease of protrusive activity at the free edges of the contacting cells. Neoplastic transformation changes cell-cell interactions, preventing contact paralysis after the establishment of cell-cell contact, leading to overlapping of lamellae and to the extension of lamellipodia at the free edges.
To correlate cell behavior with the dynamics of junctional E-cadherin, we established nontransformed IAR-2 cells and transformed IAR-6-1 cells stably expressing GFP-E-cadherin. Using time-lapse differential interference contrast (DIC) and fluorescence microscopy, we investigated living cells for 3–7 h. In nontransformed IAR-2 cells, GFP-E-cadherin accumulated in stable continuous tangential AJs at the cell-cell boundaries (Figure 5A and Video S6). When IAR-2 cells formed new cell-cell contacts, GFP-E-cadherin assembled in tangential lines along the cell-cell boundaries. In contrast, in transformed IAR-6-1 cells, AJs were dynamic (Figure 5B and Video S7). Transformed cells extended protrusions over the entire periphery. When a protrusion contacted another cell, GFP-E-cadherin began to accumulate in dot-like clusters at the site of the cell-cell contact, which were able to grow. In IAR-6-1 cells, we found the remodeling of AJs. AJs in IAR-6-1 cells could rearrange and relocate in the zone of contact. Unlike stable AJs in IAR-2 cells, AJs in IAR-6-1 cells were often disrupted. As a cell detached from one cell, it could form a contact with another cell, in which GFP-E-cadherin accumulated. Thus, transformed IAR-6-1 cells could move and establish dynamic E-cadherin-based cell-cell contacts with neighboring cells.
Using the distance map method [25], we analyzed the dynamics of AJs in IAR-2 cells and IAR-6-1 cells stably expressing GFP-E-cadherin (Figure S4). Time-lapse sequences of sparse cultures were used to calculate the mean rates of AJ movement. We found differences in the dynamics of AJs in IAR-2 cells and IAR-6-1 cells. While the mean rate of movement of tangential AJs in IAR-2 cells was 59±3 nm/min (mean±SEM, n=28), the mean rate of movement of radial AJs in IAR-6-1 cells was 189±21 nm/min (n=30). Using ImageJ software, we also measured the distances traveled by individual AJs, and calculated the mean rates of AJ movement. This analysis showed similar results: the mean rate of movement of AJs in IAR-2 cells was 97±17 nm/min (mean±SEM, n=25), the mean rate of movement of radial AJs in IAR-6-1 cells was 176±22 nm/min (mean±SEM, n=25).
To compare the dynamics of AJ assembly in nontransformed and transformed epithelial cells thoroughly, we investigated cell-cell collisions in a narrow wound. Live-cell imaging of IAR-2 cultures showed that during the establishment of the cell-cell contact, a tangential line of GFP-E-cadherin formed at the cell-cell boundary and expanded laterally in two opposite directions (Figure 6A and Video S8). The characteristics of GFP-E-cadherin accumulation at the sites of cell-cell contacts in cultures of IAR-6-1 cells were different (Figure 6B, 6C, and 6D, and Video S9). At the site of the cell-cell contact, GFP-E-cadherin initially aggregated into dot-like clusters. Some of these puncta could disappear. The majority of GFP-E-cadherin clusters grew and transformed into radial strands oriented perpendicularly to the cell-cell boundary. AJs could change their position. During cell displacement, AJs between two adjacent cells in a monolayer often elongated and broke. Thus, our studies revealed two different types of E-cadherin-based AJs: stable tangential AJs in nontransformed epithelial cells and relatively dynamic radial AJs in transformed epithelial cells.
To elucidate the mechanisms of the formation of different types of AJs and the rearrangement of tangential epithelial AJs into radial ones during neoplastic transformation, we examined the involvement of the Rho family small GTPases, Rho and Rac, in regulating cell-cell adhesion. Earlier, we had described radial AJs in Rat-1 fibroblasts associated with short straight actin bundles. As it has been shown that the formation of radial AJs in fibroblasts depends on myosin II-mediated contractility [26], [27], we decided to add comparisons of the effects of inhibition of Rho or Rac signaling on radial AJs in Rat-1 fibroblasts to our studies concerning the effects of this inhibition on radial AJs in transformed IAR-6-1 epithelial cells.
First, we investigated the effect of inhibition of Rho activity on AJ formation in nontransformed IAR-2 cells, transformed IAR-6-1 cells, and Rat-1 fibroblasts (Figure 7). C3, which ADP-ribosylates and which inactivates cellular Rho, was introduced into wound-edge cells during the wounding of monolayers. Wounding of a cell monolayer with a hypodermic needle in the presence of C3 and fluorescent dextran (as a marker) resulted in loading of ~50% cells at the wound edge. Control experiments showed that fluorescent dextran alone did not affect the rate of wound healing or the accumulation of adhesive proteins in the cell-cell contacts. Loading of cultured cells with C3 led to a delay of wound healing up to 7–8 h (3–4 h in controls). Actin bundles in C3-loaded cells were disrupted (data not shown). The introduction of C3 into IAR-2 cells prevented the formation of tangential AJs. In C3-loaded IAR-2 cells, E-cadherin did not accumulate at the cell-cell boundary. Transformed IAR-6-1 cells loaded with C3 could not assemble radial AJs. E-cadherin aggregated only in dot-like clusters. Similar dot-like clusters of N-cadherin were seen at the site of the contact between C3-loaded Rat-1 fibroblasts. These cells failed to form radial AJs. Thus, Rho activity is required for the formation of tangential and radial AJs.
To assess the impact of Rho effectors in the establishment of AJs of different types, we first compared the effect of the specific ROCK inhibitor Y-27632 on the formation of tangential AJs in nontransformed IAR-2 cells and of radial AJs in transformed IAR-6-1 cells and Rat-1 fibroblasts (Figure 8A). Addition of 30 µM Y-27632 to culture medium for 2–3 h resulted in disassembly of both marginal and straight actin bundles in IAR-2 cells. Nevertheless, in the presence of Y-27632, E-cadherin accumulated in tangential AJs that had a more wave-like shape than AJs in control IAR-2 cells. In the presence of Y-27632, the establishment of cell-cell contact was not accompanied by contact paralysis of colliding cells. Treated with Y-27632, IAR-2 cells continued to form lamellipodia at the sites of cell-cell contacts (Figure S5). In the case of transformed IAR-6-1 cells, 30 µM Y-27632 abolished actin bundles. Radial E-cadherin-based AJs did not assemble at the sites of cell-cell contacts. We saw staining of E-cadherin only in dot-like clusters. Y-27632 (30 µM) destroyed actin bundles and completely prevented the formation of radial AJs in Rat-1 fibroblasts. We were able to visualize only a few dots of N-cadherin at the sites of cell-cell collisions.
It is now well-recognized that Rho/ROCK signaling controls the creation of myosin II-driven forces. We examined the effect of the myosin II ATPase inhibitor blebbistatin [28] on the formation of tangential and radial AJs (Figure 8A). Our experiments showed that tangential contacts can assemble without myosin II activity. In the presence of 50 µM blebbistatin, which completely destroyed marginal and straight actin bundles in IAR-2 cells, E-cadherin accumulated in a wavy line oriented along the cell-cell boundary. Blebbistatin (50 µM) abolished actin bundles and prevented the formation of radial AJs in IAR-6-1 cells. In these cells, we revealed only dot-like and fragmented linear junctions at the sites of the cell-cell contacts. In Rat-1 cells, 50 µM blebbistatin had quick and strong effect on cell morphology, the actin cytoskeleton, and AJs and led to cell arborization (data not shown). In the case of Rat-1, we used a lower dose of blebbistatin (15 µM), which also destroyed actin bundles and stopped the assembly of radial AJs.
To quantify the effects of Y-27632 and blebbistatin on cadherin accumulation at AJs, fluorescence intensities of E-cadherin and N-cadherin at cell-cell contacts were measured as described previously [23]. Analysis of fluorescence intensity revealed that Y-27632 and blebbistatin dramatically reduced the accumulation of E-cadherin at AJs in IAR-6-1 cells and of N-cadherin at AJs in Rat-1 cells (Figure 8B). In contrast, we did not find significant differences between the average levels of E-cadherin fluorescence at cell-cell contacts in control and drug-treated IAR-2 cells.
The studies performed suggest that the establishment and maintenance of radial AJs both in transformed epithelial cells and in fibroblasts depend on myosin II-mediated contractility. In contrast, myosin II is not required for the assembly of tangential AJs in nontransformed epithelial cells.
To investigate the involvement of another Rho effector, mDia1 formin, in the formation of AJs, we applied the RNAi technique for the depletion of mDia1 [29]. We used two types of mDia1 siRNA duplex that had similar effects. We assayed the efficiency of suppression of mDia1 using Western blot analysis of cell lysates 48 h post-transfection. Transfection with mDia1 siRNA significantly decreased the expression of mDia1 in all three cell types (Figure 9A and 9B). For double immunofluorescence staining of mDia1 and AJs, we used E-cadherin mouse monoclonal antibodies (IgG2a isotype) and mDia1 antibodies (IgG1 isotype) revealed by secondary mouse IgG1 and mouse IgG2a antibodies. Comparison of mDia1 immunofluorescence staining of cultures revealed a dramatic decrease of mDia1 in cells 48 h after transfection (Figure 9D). Depletion of mDia1 resulted in a prominent disturbance in the formation of tangential AJs in IAR-2 cells. In all cell pairs with diminished staining of endogenous mDia1 as a result of mDia1 suppression by RNAi, tangential AJs did not assemble. At the sites of cell-cell contacts, E-cadherin could accumulate only in dot-like clusters. These data are in agreement with the results of Carramusa et al. [23] concerning the disappearance of AJs in MCF7 epithelial cells after short-hairpin RNA knockdown of Dia1. We also compared the effects of mDia1 depletion on the formation of radial AJs in transformed IAR-6-1 epithelial cells and Rat-1 fibroblasts. Unlike tangential AJs, radial AJs assembled in mDia1 siRNA-transfected cells. Double immunofluorescence staining revealed that in IAR-6-1 cells transfected with mDia1 siRNA, E-cadherin accumulated in radial AJs. Depletion of mDia1 did not influence the assembly of radial AJs in Rat-1 fibroblasts. To demonstrate this, we established Rat-1 cells stably expressing E-cadherin that was colocalized with N-cadherin in AJs (data not shown). Using anti-E-cadherin antibodies to localize AJs in Rat-1 fibroblasts and anti-mDia1 antibodies to reveal the cells transfected with mDia1 siRNA, we found that mDia1 suppression by RNAi did not prevent the formation of radial AJs in Rat-1 fibroblasts.
To assess the influence of mDia1 depletion on cadherin accumulation at AJs, we measured the fluorescence intensities of E-cadherin at cell-cell contacts on immunofluorescence images (Figure 9C). We found that the fluorescence intensity of E-cadherin in IAR-2 cells transfected with mDia1 siRNA was reduced on average by ~50% in comparison with that of the control. In contrast, we did not find significant differences between the fluorescence intensities of cadherins at cell-cell contacts in control and N17Rac-loaded IAR-6-1 and Rat-1 cells.
Thus, mDia1 contributes to the formation of tangential AJs in nontransformed IAR-2 epithelial cells but is not involved in the assembly and maintenance of radial AJs in transformed epithelial cells and fibroblasts.
Rac signaling has been shown to play an essential role in the establishment of epithelial AJs [8], [9]. We also examined a possible alteration of its contribution to the regulation of AJ assembly in transformed epithelial cells. A dominant-negative form of Rac, N17Rac, was loaded into cells during the wounding of monolayers to suppress the activity of cellular Rac (Figure 10A). IAR-2 cells loaded with N17Rac did not form continuous tangential AJs. In these cells, E-cadherin could accumulate only in dot-like aggregates or short linear fragments at cell-cell boundaries. In contrast, N17Rac did not affect the formation of radial E-cadherin-based AJs in transformed IAR-6-1 cells and of N-cadherin AJs in Rat-1 fibroblasts.
To assess the influence of N17Rac loading on cadherin accumulation at AJs, we also measured the fluorescence intensities of E-cadherin at cell-cell contacts in IAR-2, IAR-6-1, and N-cadherin in Rat-1 cells (Figure 10B). Analysis revealed that the fluorescence intensity of E-cadherin in N17Rac-loaded IAR-2 cells was reduced on average by ~50% in comparison with that of the control. In contrast, we did not find significant differences between the fluorescence intensities of cadherins at cell-cell contacts in control and N17Rac-loaded IAR-6-1 and Rat-1 cells. Thus, the assembly of tangential AJs required Rac activity. In contrast, radial AJs in transformed epithelial cells and fibroblasts did not depend on functions of Rac. Collectively, the functional studies demonstrated the similarities in regulation of assembly of radial AJs in transformed epithelial cells and radial AJs in fibroblasts by Rho family small GTPases.
This study is the first to demonstrate that transformed epithelial cells can use the disappearance of the marginal actin bundle and the remodeling of E-cadherin-based AJs to weaken cell-cell adhesion and to acquire motile behavior and migratory activity. When we studied transformed IAR-6-1 and IAR1170 epithelial lines, we found that while cells of these lines retain E-cadherin expression, have a morphology slightly changed from the morphology of the parent nontransformed IAR-2 cells, and form monolayers in confluent culture that are similar to IAR-2 monolayer, immunofluorescence staining demonstrated dramatic rearrangements of the actin cytoskeleton and AJs. In transformed IAR-6-1 and IAR1170 cells, the adhesion belt is broken and replaced by E-cadherin-based puncta or strands resembling radial AJs of fibroblasts. AJs in transformed epithelial cells are associated with short straight actin bundles. Our data demonstrated that in contrast with stable tangential AJs in nontransformed epithelial cells, radial E-cadherin-based AJs in transformed cells are very dynamic.
To elucidate the mechanisms of formation of tangential and radial AJs, we next analyzed the involvement of small Rho GTPases and their effectors in AJ assembly. Our functional studies revealed the differences in the main driving forces of AJ formation in nontransformed and transformed epithelial cells. We found that mDia1 and Rac signaling are essential for the assembly of tangential AJs only in nontransformed epithelial cells. In contrast, Rac activity and mDia1 are not required for the assembly of radial AJs in transformed epithelial cells and fibroblasts. Earlier, Braga et al. [30] proposed that E-cadherin may be regulated by Rac differentially depending on cellular background (physiological context). Our data demonstrate the involvement of Rac signaling in the establishment of cadherin-mediated adhesion depending on the spatial organization of AJs. We propose that these differences are related to differential regulation of actin polymerization in the areas of tangential and radial AJs. Regulation by Rac1 and mDia1 of actin polymerization in tangential AJs is important for their establishment and stability.
We also showed that ROCK-stimulated myosin II activity is not required for the formation of tangential AJs in nontransformed epithelial cells. These findings confirmed the data of Sahai and Marshall [21] and Ivanov et al. [12], which established that myosin II motor activity is not essential for AJ assembly in epithelial MDCK and T84 cells. In contrast, myosin II-mediated contractility is a key regulator of the formation of radial AJs in transformed epithelial cells. As we and others discovered earlier, the formation of radial AJs in fibroblasts depends on myosin II-mediated contractility [26], [27]. Radial AJs in transformed epithelial cells are very similar to radial AJs in fibroblasts. Our study involving live-cell imaging of GFP-E-cadherin in transformed epithelial cells showed that AJs initially appear as dot-like clusters that grow into puncta or radial strands. We propose that the maturation of initial cadherin-based dot-like clusters into radial junctions depends on (i) association of an adhesion plaque with actin filaments, (ii) myosin II and α-actinin cross-linking of actin filaments into straight actin bundles, and (iii) centripetal myosin II-mediated tension of straight actin bundles, leading to growth of radial AJs. We also propose that, regulated by mechanical forces, actin polymerization like the recently described force-dependent actin polymerization in focal contacts [31] plays an important role in the formation of radial AJs. Local balance between myosin II-mediated contractile forces, adhesive interaction of cells with neighboring cells, and the generation of traction in the direction of lamellipodia extension contribute to growth, adhesion strengthening, and disruption of radial AJs.
We hypothesize that the rearrangement of AJs in epithelial cells during neoplastic transformation into radial AJs is defined by reorganization of the actin cytoskeleton, leading to alterations of directions of tension in the area of cell-cell contacts. As we found earlier [32], the formation of tangential E-cadherin-mediated cell-cell contact in nontransformed epithelial cells leads to disruption of marginal bundles at the site of contact and to inhibition of retrograde actin flow. The remaining segments of marginal bundles of adjacent cells form two arc-like bundles (Figure 11) containing myosin II [33], which produce tangential tension in the zone of cell-cell contact in nontransformed epithelial cells. In transformed epithelial cells, disappearance of the marginal actin bundle is a key event that results in the rearrangement of AJs. In these cells, the deficiency of tangential tension and centripetal tension in the zone of cell-cell contact leads to the assembly of straight actin bundles and to the maturation of radial AJs.
In the present study using live-cell imaging, we also revealed significant alterations of cell-cell interactions caused by neoplastic transformation. The formation of stable contact between nontransformed epithelial cells results in dramatical inhibition of protrusions at the site of contact (contact paralysis) and in a decrease of protrusive activity at the free edges of contacting cells. In contrast, contact paralysis is not seen in transformed epithelial cells. Analysis of kymographs showed that lamellipodia at the sites of the cell-cell contacts continued to extend. Transformed cells also form lamellipodia at the free edges. We suggest that tangential tension of arc-like bundles in nontransformed epithelial cells may decrease the extension of protrusions both at the site of the cell-cell contact and at the free edges of contacting cells. Inhibition of protrusive activity stabilizes the contact between two cells. Similar suppression of protrusive activity after the application of artificial tangential tension with a micromanipulating needle was described by Kolega [34]. Tangential tension also can cause fast lateral expansion of contact and align newly assembled actin filaments in perijunctional bundles, strengthening the adhesion. The role of marginal actin bundles in the creation of tangential tension and the decrease of protrusive activity of contacting nontransformed epithelial cells was also supported by our experiments with Y-27632 and blebbistatin, which abolish marginal bundles. In the presence of these inhibitors, contact paralysis during the formation of cell-cell contact was not developed. We propose that the absence of marginal bundles in transformed epithelial cells leading to the deficiency of tangential tension at the border of contacting cells prevents inhibition of protrusive activity of contacting cells. The experiments performed suggest that the alterations in cell-cell interactions and the remodeling of AJs change motile behavior and migratory activity of transformed epithelial cells.
It should be noted that the cell-cell interactions between transformed epithelial cells described in this paper are very similar to those between cells of mesenchymal origin (fibroblasts). Earlier, we described the absence of contact paralysis and significant overlapping between contacting fibroblasts followed by persisting formation of lamellipodia at the free edges [26]. The formation of E-cadherin-based AJs in transformed epithelial cells and their regulation by Rho GTPases is similar to those in fibroblasts. Possibly, these alterations in transformed epithelial cells are manifestations of the initial stages of EMT. This question obviously needs further investigation.
Thus, our studies demonstrate that structural and dynamic coordination between cadherins, actin structures, and actin-regulating proteins can change during neoplastic transformation. In summary, we suggest a hypothetical scheme for the rearrangements of AJs and the actin cytoskeleton at the early stages of morphological transformation (Figure 11). These alterations are initiated by disruption of the marginal actin bundle. The deficiency of tangential tension as a result of the disappearance of the marginal bundle prevents contact paralysis and leads to overlapping of contacting cells. Dot-like AJs form in the zone of overlapping. Centripetal myosin II-mediated tension converts them into radial AJs, which are very dynamic and less stable than tangential AJs in nontransformed epithelial cells. Changes in the activities of Rho family small GTPases, which are characteristic of transformed cells, can be involved in these rearrangements. Remodeling of AJs and alterations of protrusive activity modulate cell-cell adhesion and motile behavior of cells. It is tempting to speculate that rearrangements of the actin cytoskeleton and of E-cadherin-based AJs observed in the present study may be essential for carcinoma cell dissemination.
The ROCK inhibitor Y-27632 and the myosin II ATP-ase inhibitor (±)-blebbistatin were purchased from Calbiochem-Merck. The following primary monoclonal antibodies were used: anti-E-cadherin, clone 36; anti-N-cadherin, clone 32; anti-mDia1, clone 51 (Transduction Laboratories-BD); and anti-α-tubulin, clone DM 1A (Sigma-Aldrich). Secondary TRITC-conjugated antibodies were obtained from Chemicon; secondary Alexa488-conjugated antibodies; and Alexa488- and TRITC-conjugated phalloidin were obtained from Molecular Probes-Invitrogen. Secondary anti-mouse IgG1 and IgG2a antibodies were purchased from Southern Biotech. Horseradish peroxidase-conjugated secondary antibodies for Western blotting were purchased from Upstate-Millipore. Other reagents were obtained from Sigma-Aldrich.
The IAR-2 line of nontransformed rat epithelial cells and the IAR-6-1 line of IAR epithelial cells transformed with dimethylnitrosamine were established by Montesano et al. [35]. The pPS/hygro-N-RasG12D construct was obtained from B.P. Kopnin (N.N. Blokhin Cancer Research Center, Russia). We obtained the IAR1162 and IAR1170 lines, expressing N-RasG12D, using retroviral infection from IAR-2 cells according to the procedure of retroviral transduction and selection with hygromycin [36]. We established individual clones of Ras-expressing IAR1162 cells (nine clones) and IAR1170 cells (five clones). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a 5% CO2 humidified atmosphere. E-cadherin- and GFP-E-cadherin-expressing constructs were kindly provided by Dr. S.M. Troyanovsky (Northwestern University, Chicago, USA). We stably transfected Rat-1 fibroblasts with E-cadherin plasmid using the FuGene6 transfection reagent according to the manufacturer's protocol (Roche). To generate IAR-2 and IAR-6-1 lines that stably expressed GFP-E-cadherin, we transfected IAR-2 and IAR-6-1 cells with GFP-E-cadherin construct using the FuGene6 reagent. Stable transfectants were established after 2 weeks of selection in medium with G-418 (1 mg/ml).
Cell-cell collisions and the establishment of cell-cell contacts were investigated in narrow wounds. We created narrow wounds by scraping cell monolayers on coverslips with an injection needle. Cultures were incubated for 3–4 h and observed or fixed at the time when the wound started to close. Y-27632 and blebbistatin were added to the growth medium from stock solutions at 20 min after wounding.
To evaluate the effects of Rho and Rac inhibition on the establishment of cadherin-mediated AJs in a narrow wound, C3 transferase and dominant negative N17Rac (kindly provided by Dr. A. Hall, Memorial Sloan-Kettering Cancer Center, USA) were used. Recombinant proteins were prepared as glutathione S-transferase fusion proteins in Escherichia coli, purified with glutathione Sepharose beads, thrombin cleaved, dialyzed against microinjection buffer, and concentrated as described previously [37]. C3 transferase and N17Rac were introduced into wound edge cells at the time of wounding, as a modification of the method of Brock et al. [38]. TRITC-labeled 10,000-MW lysine-fixable dextran (Molecular Probes) was used as a marker of loaded cells. Coverslips with cell monolayers were transferred into new dishes. Aliquots (10 µl) of protein solution (0.5 mg/ml) mixed with 1 µl of dextran were added on the monolayer surface. Monolayers were wounded with an injection needle, rinsed with warm DMEM, and incubated in DMEM before fixation with 3.7% paraformaldehyde (PFA).
To deplete mDia1 in cells, we used the RNAi technique. The small interfering RNA oligonucleotides, corresponding to two mDia1 sequences from positions 795 to 813 and 184 to 209, were described previously [29] and chemically synthesized by Syntol (Moscow, Russia). Synthetic siRNA duplexes based on sense and antisense sequences were prepared as described [39]. GFP siRNA was used as negative control. We transfected mDia1 siRNA or control siRNA duplex oligonucleotides into cells using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). The cultures were incubated for 36–48 h and analyzed with Western blotting or fixed after wounding of cell monolayers and establishment of new cell-cell contacts.
We determined the migratory capacity of IAR cells in vitro using BD Bio-CoatTM migration chambers containing polycarbonate membrane inserts with 8-µm pores in a 24-well tissue culture plate (Becton Dickinson). Cells (2×104 cells/200 µl of DMEM with 8% FCS) were added in the upper wells of chambers. The bottom well of each chamber was filled with DMEM with 8% FCS. After 20 h of incubation at 37°C with 5% CO2, cells from the upper surface of membranes were completely removed with a cotton swab; the migratory cells on the lower surface of membranes were fixed with 100% methanol and stained with DAPI. Membranes were mounted onto glass slides and examined microscopically at 20× magnification. We determined cellular migration by counting the numbers of stained cells on membranes in 15 randomly selected fields. The means of triplicate assays for each cell line in three independent experiments were used.
Cells were resuspended in DMEM (1 million cells/300 µl DMEM) and were injected subcutaneously into the right rear flank of athymic nude (nu/nu) mouse (eight mice per group). Animals were monitored weekly for appearance and measurement of tumors. Tumor volumes (V) were determined from the three dimensions (height (a) by width (b) by depth (c) of tumors) (in millimeters) and calculated as V=1/6πabc according to [40]. Animals were sacrificed 4 weeks postinjection, and the tumors, where present, were analyzed histologically and weighed.
For E- or N-cadherin staining, cells were fixed with methanol-acetone (11) at −20°C for 10 min. For double staining of E- or N-cadherin and of actin, cells were fixed with 1% PFA and permeabilized with 0.5% Triton X-100 for 5 min. To visualize mDia1, cells were fixed with 3.7% PFA and permeabilized. In some experiments, fixed cells were heated to 100°C and transferred into fresh PBS. Fixed specimens were incubated for 40 min with primary antibodies and then for 40 min with TRITC- or Alexa488-conjugated secondary antibodies. Alexa488- or TRITC-conjugated phalloidin was added to secondary antibodies. Fixed samples were examined with a Zeiss Axioplan microscope equipped with a 100× (NA 1.4) objective. Images were acquired with a cooled CCD ORCA-ER camera (Hamamatsu Photonics) using Wasabi 1.5 software.
We measured the intensity of E-cadherin and N-cadherin fluorescence at cell-cell contacts using ImageJ software (NIH, USA) as described [23]. The average of peak pixel intensity values in square segments (10×10 pixels) covering the entire cell-cell contact length was taken as a measure of fluorescence intensity at cell-cell contacts. For the quantification of cadherin accumulation at cell-cell contacts, we calculated a localization index by subtracting the peak value of cytoplasmic intensity from the peak value at the cell-cell contact and dividing this difference by the peak value of cytoplasmic intensity. This index was used to calculate relative fluorescence intensity. Control values were taken as 1.
For live-cell imaging, cells were transferred into phenol red free α-MEM medium (Gibco, Invitrogen) supplemented with 10% FCS and were observed with a Nikon Eclipse-Ti microscope equipped with a Plan-Neofluar 100× (NA 1.3) DIC objective at 35°C. Images were acquired with an ORCA-ER Hamamatsu camera controlled with NIS-Elements AR2.3 software (Nikon). In some cases, we used Leica TCS SP5 confocal laser scanning microscope. We measured overlapping areas as described [32], using SimplePCI 5.3.1 software (Hamamatsu). To analyze lamellipodial dynamics in the narrow wound, time-lapse sequences were acquired with 3-s intervals for 25–30 min. Using ImageJ software (NIH, USA), we generated kymographs along straight lines drawn in the direction of protrusion. Rates of lamellipodial protrusion and retraction were determined on the basis of slopes produced by advancing leading edges. The mean rates during 5 min periods of observation were used.
Using ImageJ software, we measured the distances traveled by individual AJs and calculated the mean rates of AJ movement. The distance map method was used to study the dynamics of AJs. We analyzed selected frames from confocal images of GFP-E-cadherin in living cells in sparse cultures using ImageJ software as described [25]. Binary images produced from grey images were thinned until they formed binary skeletons, which represent central positions of AJs. Distance maps (Di) were created; these are images of the distance to the nearest point from the skeleton. Next, the binary skeletons at times i+1 were overlaid on the distance maps (Di). In the absence of movement, the pixel value on the distance map below the skeleton is zero. If movement occurred between the two images, the pixel value below the skeleton indicates the distance traveled between times i and i+1. The average value of the pixel-to-pixel multiplication of Si+1 and Di quantifies the average displacement of AJs between two successive images.
SDS-PAGE and Western blotting were performed in minigel PAGE devices (Mini-Protean® 3 Cell, Bio-Rad). For Western blotting of whole cell lysates, cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 16% glycerol; 1% mercaptoethanol; 0.004% bromphenol blue) including protease inhibitor cocktail (Sigma). Samples (10 or 20 µl) were heated for 5 min at 95°C and used immediately for 10% SDS-PAGE in equal protein concentrations according to the Bio-Rad protocol. Resolved proteins were transferred to PVDF membranes, which were then probed with appropriate primary and peroxidase-conjugated secondary antibodies. We detected blotted protein bands using an ECL Plus kit (Amersham), and we captured chemiluminescence images using an image acquisition Chemi-Smart 2000 system (Vilber Lourmat) and Chemi Capt software.
Expression of E-cadherin and N-cadherin in nontransformed IAR-2 cells and transformed IAR-6-1, and IAR1170 cells. Total cell lysates were resolved by means of SDS-PAGE and immunoblotted for E-cadherin (A), N-cadherin (B), and α-tubulin (as loading control). Nontransformed IAR-2 cells express epithelial E-cadherin. Transformed IAR-6-1 cells express E-cadherin and do not express N-cadherin. Transformed IAR1170 cells retain E-cadherin and also express N-cadherin.
Tumorigenic effects of the injection of transformed IAR-1170 cells (clone H5). Cells were injected subcutaneously into athymic nude mice. (A) Tumor volume was measured as described in Materials and Methods. (B) Tumor weight was determined at 4 weeks postinjection, when animals were sacrificed. Each value is the mean±SEM of eight determinations. Tumors were not formed after injection of nontransformed IAR-2 cells.
Kymographs generated at the free edges of contacting cells (left) were used to quantifiy the rates of lamellipodial protrusion and retraction (right). In nontransformed IAR-2 cells, the establishment of cell-cell contacts resulted in the decrease of the rates of lamellipodial protrusion at the free edges (from 4.0 µm/min to 2.6 µm/min) (***, p<0.05, t-test). In transformed IAR-6-1 cells, the rates of lamellipodial protrusion and retraction at the free edges of contacting cells did not change within the time of observation. White line on kymograph indicates the time of the establishment of the contact. Data are presented as means±SEM.
Schematic representation of the distance map method for measurement of the rate of AJ movement. (A and B) Fragments of confocal images of AJs at times i and i +3 min (image size, 34 µm). Images were acquired at 3-min intervals. (C and D) Skeleton images corresponding to images A and B. (E) Map of the distances from the skeleton at time i. (F) Result of the multiplication of images D and E. The intensity of each pixel in image F corresponds to the distance in pixels traveled by AJs during 3 min.
In the presence of Y-27632, the establishment of the contact between IAR-2 cells is not accompanied by contact paralysis. Left: kymograph generated at the site of the cell-cell contact. White line indicates the time of the establishment of the contact. Right: quantification of the rates of lamellipodial protrusion and retraction at the sites of cell-cell contacts.
Stable cell-cell contacts in an IAR-2 island. Cell-cell contacts are stable during the entire period of observation (Re: Figure 2A). Images were acquired at 5-min intervals for 195 min.
Cell-cell interactions in a sparse culture of transformed IAR-6-1 cells. Cells extend lamellipodia at different sites along free edges and establish contacts with different neighboring cells. Cell-cell contacts are unstable and often break. Cells move in different directions (Re: Figure 2B). Images were acquired at 5-min intervals for 360 min.
Cell-cell interactions in a sparse culture of transformed IAR1170 cells (clone H5). The cells establish dynamic contacts with neighboring cells. Cell-cell contacts are unstable and may break (Re: Figure 2C). Images were acquired at 5-min intervals for 425 min.
Dynamics of cell-cell interactions between nontransformed IAR-2 cells in a narrow wound. By 15-20 min, protrusive activity at the contact site is inhibited (contact paralysis). At the same time, the extension of lamellipodia at the free edges of contacting cells decreases (Re: Figure 4A). Images were acquired at 3-s intervals for 20 min.
Dynamics of cell-cell interactions between transformed IAR-6-1 cells in a narrow wound. Upon formation of the contact, protrusions extend both at the site of the contact (absence of contact paralysis) and at the free edges (Re: Figure 4C). Images were acquired at 15-s intervals for 28 min.
GFP-E-cadherin dynamics at the sites of cell-cell contacts in a sparse IAR-2 culture. Nontransformed IAR-2 cells were stably transfected with GFP-E-cadherin. GFP-E-cadherin accumulates in the zones of cell-cell interactions, forming stable tangential AJs (Re: Figure 5A). Images were acquired at 10-min intervals for 170 min.
Remodeling of AJs in transformed IAR-6-1 cells. Transformed IAR-6-1 cells were stably transfected with GFP-E-cadherin. Upon establishment of cell-cell contact, E-cadherin aggregates in dot-like AJs at the site of the contact. AJs can grow, rearrange, and relocate in the area of the contacts. The disruption of AJs results in detachment of one cell from another (Re: Figure 5B). Images were acquired at 10-min intervals for 320 min.
Accumulation of GFP-E-cadherin at the contact between two nontransformed IAR-2 cells in a narrow wound. During the formation of the cell-cell contact, GFP-E-cadherin accumulates in a tangential line at the boundary between two cells (Re: Figure 6A). Images were acquired at 30-s intervals for 13 min.
GFP-E-cadherin dynamics at the site of the cell-cell contact of transformed IAR-6-1 cells in a narrow wound. GFP-E-cadherin initially accumulates in small, dot-like clusters. A majority of E-cadherin dots transform into radial strands. Within the time of observation, the size of radial AJs can grow (Re: Figure 6B). Images were acquired at 30-s intervals for 47 min.
The authors thank Dr. Sergey M. Troyanovsky for providing E-cadherin constructs. We also thank Natalya Chromova for providing nude mice and Dr. Tatyana G. Moizhess for help in mouse tumorigenicity analyses. We thank Dr. Svetlana Rubtsova for help with software and for critical reading of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was funded from the Russian Foundation for Basic Research (Grant 08-04-00408a). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Miracle on the 17th Green Trailer (1999)
19 December 1999 100 mins
A 50 year old adman (Robert Urich) loses his job. Rather than facing trying to find a new job, he decides to try to make it on the senior golf tour. This causes him to neglect his wife (Meredith Baxter) and family.
Watch the official Miracle on the 17th Green 1999 trailer in HD below or find more Miracle on the 17th Green videos on Vidimovie.

subset: commoncrawl


text:
Sold by Suzanne Dingley. Must-see home! Outstanding remodeling job done by architect/owner. Soaring ceilings with 2-story brick fireplace, morning room, screened porch. 1st floor office plus bedroom with full bath. Nicely finished walk-out basement has rec room with exercise area. Beautiful paver sitting area in back yard. Lovely private setting in Robinwood Estates. Convenient location. Shenendehowa Schools: Orenda Elementary, Gowana Middle.
Listing information
- Year Built: 1983
- Status: Sold on 9/16/2013
- Fireplaces: 1
- Foreclosure: No
- Lot Area: 0.83
- County: Saratoga
- Elementary School: Orenda
- Subdivision: Robinwood Estates
- Property ID: 102866395
- Beds: 4
- Total Rooms: 9
- Parking Type Description: Attached 3 cars
- Total Square Footage: 3,144 ($141.48 per sq. foot)
- Lot Size: .83 ac
- High School: Shenendehowa
- Neighborhood: Robinwood Estates
- Style: Contemporary
- Baths: 3 (Full: 3 3/4: 0 1/2: 0 Other: 0)
- Stories: 2
- New Construction: No
- Below Ground Sq. Feet: 200
- Estimated Annual Taxes: $8,791
- Middle School: Gowana
- School District: Shenendehowa
Amenities Built In Cabinets, Ceramic Tile, French Door, Gourmet Kitchen, Skylight, Vaulted Ceiling, Walk In Closet, Wall To Wall Carpet, Wood Floors
Appliances Cook Top W Grill, Dishwasher, Disposal, Microwave, Single Wall Oven
Attic Hatch, Storage
Basement Finished Area, Full, Rec Room
Condition Superior
Cooling Type Central Air
Exterior Cedar
Exterior Features Deck, Lighting, Patio, Porch Screened, Sprinkler In Ground
Garage Description Attached
Heat Fuel Natural Gas
Heat System Hot Air
Interior Features Air Cleaner, Cable Hook Up, Central Vacuum, Garage Door Opener, Gran Solid Surf Counters, High Speed Internet Acc, Humidifier, Paddle Fan, Security System, Water Softener
Lot Description Landscaped, Privacy, Treed Wooded
Master Bedroom Second Floor, Suite
Other Fuel Cost - 172, General Taxes - 1934, Lot - .83 Ac, Model - Contemporary, School Taxes - 6857, Special Assessment - Star
Other Rms Fireplace Type Fire Place Wood
Roof Asphalt Shingle
Sewer Public
Sign Yes
Survey Yes
Water Public
Rec Rm Basement
Bathroom 2nd Floor
Bedroom 2 2nd Floor
Master Bathroom 2nd Floor
Master Bedroom 2nd Floor
Bathroom 1st Floor
Bedroom 3 1st Floor
Bedroom 4 1st Floor
Dining Room 1st Floor
Great Rm 1st Floor
Kitchen 1st Floor
Office/Den 1st Floor
Listed by Suzanne Dingley of Coldwell Banker Prime Properties


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Tuesday, January 26, 2016
A Piece of Art That Oozes StarSlinger Inspiration
I found the above piece of art on Deviant Art. Western and alien at the same time. Evocative. Damn but its a good piece. It oozes StarSlinger.
I may just need to reach out to the author and see how much he wants for the rights to use it.
In any case, I do need to stat out this riding beast ;)
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|01 Oct 2013
Windows 7 Home Premium x64
Error while trying to install Office 365
While trying to install office 365, two error messages pop up. The first one is:
The application was unable to start correctly (0xc0000142). Click ok to close the application.
The message above refers to the application perfboost.exe.
The next error message is exactly the same as the previous one, only this time the application that failed to start was FIRSTRUN.EXE.
I have already uninstalled pervious office suites through the control panel, by using the Microsoft Fixit application and manually, with no avail.
Can anyone help me out on this?
Thank you in advance.
|My System Specs
|10 Dec 2013
Windows 7 Professional 64bit
I have exactly the same problem. Did you by any chance manage to fix it?
|My System Specs
|10 Dec 2013
Windows 7 Home Premium 64 bit sp1
Welcome to Seven Forums.
Try this link to see if this helps. http://support.microsoft.com/kb/2827031
Read through the information & try the Mr Fixit as well.
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text:
SearchLight iOS 10.2: The Best System Search and Apps Launcher for iPhone, iPad – SpotLight Alternative…
SearchLight Cydia tweak is developed for searching anything from system and quickly launch searched app or tweak. Its a close alternative to OS X Spotlight feature.
SearchLight is one of the best jailbreak app to search system wide settings, apps or tweaks and allows user to quickly launch whatever you have searched.
The developer "Twodayslate", has pushed an update for iOS 10.2 that brings couple of new features and capability to handle iOS 10.2 for iPhone, iPad and iPod Touch.
SearchLight is available on Cydia Store on BigBoss repository which costs you $2.99 for all future update and lifetime subscription.
Once installed, a new penal would be added to Settings app that allows you to configure this tweak for various parameter. User can use Activator gesture for the SearchLight Cydia tweak.
Lets share your thoughts, if you already using it…


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Three secrets of female psychology
These, mysterious and unique, gentle and tender, sometimes haughty and unapproachable, weak and wonderful, so different woman.
I'll start with one of my favourite parables that the Chinese sages have given the world. "... Once a man came to God and said, he was bored, God thought: from what to create a woman, since all the material went into the manufacture of men. But, he could not refuse, and set to work! He took a few rays of the sun, pensive melancholy of the moon, awe of a doe, affectionate look of chamois, beauty of a swan, slenderness of a reed, meekness of a dove, the sweetness of honey, softness of down, ease of air, and freshness of water ... mixed it all ... But in order to avoid excessive sweetness, added: impermanence of the wind, the cunning of a fox, venom of a snake, greediness of a shark, cowardice of a hare, cruelty of the tiger, tearing clouds, talkativeness of magpies, and all the horrors of the elements ... Out of all this mixture came, BEAUTIFUL WOMAN! God breathed into her soul, and gave to man and said, "Take care of her, she will not be repeated! ..." How to understand a woman? What do women want? Throughout his life and fruitful work of the famous psychotherapist Sigmund Freud did not find the answer to this question. Good and well! Would have been interesting if we were men? Female psychology is no less complex and interesting than mens. This stunning multi-faceted world of passions and emotions is not the combined world of feelings and moods, understand that it may not always be the woman herself. Well, try to figure it out together.
Today I will reveal to you three secrets of female psychology.
Secret One: About communication. For women it is above all love, communication, beauty, the world of human relations. Our sense of self and confidence is determined by our feelings and the quality of relationships. A woman will not have a full feeling of self-realization without the ability to communicate, to share their feelings and experiences with others.
How to use this knowledge?
Tips for Women:
• Knowing this secret, you should not reduce your communication to a minimum or even abandon it, even if you have a relationship with a young man or you got married.
• Communicate with a group of your friends together!
• Take time to talk on the phone. Let them not be lengthy, but they will still be in your life.
• Speak to your partner about issues of your free time and when you can meet with friends or family independently.
Tips for Men:
• No matter how much you do not like your girlfriend or wife, in any case, do not limit her to communicate with her friends. One of the most common mistakes made when the young man begins to choose, with whom his girlfriend or wife can communicate with and who she cant. The more you try to control and reduce a womans social circle, the more unhappy you are going to make her.
• Even if you do not like one of her girlfriends, let her decides who she wants to communicate with. Sometimes friends can speak ill of you, and you begin to fear that it might affect your relationship. Believe me, if a girl, listens to their opinions, and is still with you, then with time, she will reduce her communication with that friend.
• If you restrict her social circle, it will mean that you need fill it with something. Your mere presence is exactly what will not succeed! This is the feature of the female psyche: the more people she manages to discuss what she cares about, (even the trifle), the easier her life will be.
Secret Two: About emotional dips
Regardless of the occurrence of menstrual cycle every woman once a month experiences a strong emotional failure. From the outside it looks like this: You were smiling yesterday, and extremely happy. Today you woke up and think that clearly something is wrong. The world around you is not so bright, you're not so beautiful, your husband looks different, lost confidence in yourself and your own abilities ... And an enormous number of differences, BUT... in a state of emotional failure woman become irritable, hyper sensitive, depressed and tearful. It is especially hard for men to understand mood swings. Remember that emotional failures occur in every woman's life. It's okay! This is the feature of female psychology.
How to use knowledge?
Tips for Women:
Think about it, this state of emotional failure lasts 1-2 days. So it will soon pass.
• Try not to lose your temper with your loved ones.
• Think about how you will place yourself in a state of rest. For example: to lie in bed, drink a cup of hot tea, soak in the bath, go shopping, take care of yourself, etc. Fill this gap. Incidentally, it helps many women to view melodramas or soap operas. You may like to share a part of the emotional stress with the television screen.
Tips for Men:
• Stock up on care and patience :)
• During such moments be aware that your increased attention towards your loved one may be receive or rejected. Make a cup of tea, or run a hot bath. Be constant in your love and care, and if she requires space and solitude let her have it. Remember this will only last for a day or two.
• Remember that in a state of emotional failure women are very sensitive to the wording in phrases and sentences. Therefore, eliminate the guesswork from conversations or hints of her condition, as well as valuable tips on how to help her.
Secret Three: About the feeling of love and caring
Woman, to feel happy, need to feel the love and care on the part of men.
How to use knowledge?
Council for Women:
• Dear girls, stop chasing your career and try to have time to do everything you want! Remember that only the presence of a beloved man next to you will make you truly happy! Do not delay your personal life for later.
Advice for men:
• Always remember one simple phrase from the parable, I have described: "Take care of her, she will not be repeated!"
Enjoy who they are because they ARE - WOMAN! Open your own boundaries and the possibilities! It's so nice!


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(Archived document, may contain errors) THE COMMUNIST BLOC: TRANSFORMATION IN PROGRESS by Rafal H. Krawczyk November 1988 brought the 71st annive rsary of the October Revolution and the Russian adoption of the communist system. After World War H the -communist system spread around the world, completely changing the life of many nations. Now, there are almost 1.5 billion people living under the nome n klatura system and working daily under conditions of centrally planned economies. However, after decades of expansion and economic growth, all communist countries are now approaching, more or less quickly, very serious multilevel crises of which the econo m ic one looks the most disastrous. There is more and more awareness in the West that something very important is developing in the whole communist world. If the West wishes to influence this process, it is very important to understand these developments pr o perly. One can read frequently in Western newspapers and periodicals the thesis summed up by Jackson Diehl's article in 77ze Washington Post of October 16, 1988: "Communist World Can't Jump Price Reform Hurdle." Ile article is quite typical of Western opi n ion on the economic troubles that have plagued the communist world since the beginning of the 1980s. In Poland this year the inflation rate will probably exceed 100 percent. In Yugoslavia, the rate is more than twice that. China faces unexpected obstacles in her economic reform, and the Chinese government, frightened by an inflation rate of 50 percent annually (the highest since the communists took power) is slowing the implementation of new measures. In the Soviet Union, the future of economicperestroika l ooks gloomy. Jumping the Price Hurdle. All of the evidence seems to support the title of Diehl's article. Other Western evaluations are very similar to Diehl's. 77ze New York 7-imes of October 14 states that "without an end to subsidies and establishment o f market prices, which means a painful interim of austerity and inequality, there is no way to economic health." It does not say, however, how to establish market prices if the market simply does not exist in communist countries. It is also worth adding t h at most reformers in centrally planned economies are eagerly accepting the approach of the international financial community, which dictates that, to revive economic growth, a country must first raise consumer prices in order to diminish state subsidies. T hus, communist governments list 4"price reform" as the first objective on their agenda. Ile question arises, however: why can't they succeed in jumping the price reform hurdle despite repeated efforts, if it follows its own beliefs and Western advocates? Could there be some trap hidden behind the hurdle?
Rafal H. Krawczyk is a Bradley Resident Scholar at The Heritage Foundation, onleave from the Catholic University in Lublin, Poland. He spoke at The Heritage Foundation on November 3,1988. ISSN 0272,1155. 01989 by The Heritage Foundation.The facts speak for themselves. Yugoslavia introduced her economic reform based on the idea-of "self-governing" socialism and free market prices in 1965. Hungary started her effort to reach a "socialist controlled market" ultimately in 1968. China introduced reform in 1978, widely recognized as the world's most dynamic transformation from a Stalinist economic model to a free market. Poland, after the convulsive years of Solidarity and martial law, climbed two "stages" of e conomic reform. The first one began in 1982 with 300 percent price hikes. Ile second one started at the end of 1987 and took observers by surprise because it in fact rejected -many communist economic dogmas. It seems, however, that all these efforts faile d to achieve their objectives, and the communist world has experienced repeated waves of reforms that simply do not work. The question is why? Using Western Criteria. It is not easy to answer this question in the United States. The American public, bankers , and journalists seem to forget sometimes about the substantial differences between the two economic systems. They try to explain developments in the East usually by painting pictures with Western brushes and Western imagination. Such a method of explanat i on does not help people in the West to understand the essence of perestroika or the sudden delay in Chinese reforms. It makes the picture dimmer instead of clearer. Understanding the East is not necessarily an American problem until the day when great bus i ness and political opportunities open up, which is not as far away as many people think. I insist that there is a great margin of probability that there will not be a single communist state on the Earth by the end of this century. Is the West prepared to b enefit from this entirely new situation? Are the reasons behind the inevitable fall of "Me Eastern Empire" properly understood in order to exploit the opportunities for world peace? What are and what will be the basic mechanisms of transformation from a S o viet-style economy to a free market one? Is a free market transformation a prerequisite to democracy? These are questions.which, if improperly answered, may bring about revolutionary and violent developments, harming not only the one-third of the world po p ulation living under the communist system, but the Free World itself. Communism is a creature walking on four legs: ideology, a "flat social structure," a centrally planned economy, and a political nomenklatura system. All of these have a very practical m e aning, and they were very useful and even efficient in the past. All of them must work together to make the system energetic. But now, after decades of militancy based on the potent aggregation of most of the national resources under control of the state, all four legs apparently have become more or less consumptive. All four legs are shaking, and walking seems to be more and more painful. IDEOLOGY The practical meaning of ideology is hope. People's expectations of a better future (maybe not for themselve s but surely for their children) had been in the early stages of communism one of the most important incentives 'for hard work and "mass political activity." However, the future has already arrived, and communist nations and their political activists face empty shops and long lines of tired people hoping to buy merely the most basic goods. Instead of a promising future, communist societies have to cope with deep economic, social, and political crises. 'Me most widespread feeling among co mmunist
2nation s today is a lack of hope in a better future. That means the irreversible end of ideology. Vitali Korotich, who edits the politically lively Soviet magazine Ogonyok, when asked what he wants, what he is fighting so hard for, simply says: "I'm tired of lab e ls. I want a normal country." In Budapest, a young journalist says in disgust: "Nothing works here the way it's supposed to in a normal country." In Poland, the Solidarity leader Lech Walesa says the real issue is no longer reforms, "It's how to get out o f this abnormal system that can only produce absurdity." In the spring of 1988, Polish Prime Minister Mieczyslaw Rakowski, then a Communist Party Politburo member, distributed a secret memorandum to the communist power elite warning that the future of the S oviet Union and the communist system was in doubt. "Let's do something now before it's too late," said Rakowski. The Kremlin's new ideology chief, Vadim Medvedev, says modestly: "We have to understand better the practice of modem social democracy, and our ideas on economic and social bases of socialism need to be seriously renewed and deepened." There is no need to add to these quotations. This is the real end of the communist ideology. What can replace it? A FLAT SOCIAL STRUCTURE Social structure was flattened at the very beginning of the communist system. To be wealthy was equal to being an "enemy of the people." The top portion of society was physically or economically eliminated. The social hierarchy was turned upside down. The huge bureaucracy was recruited mainly from the bottom of society. That was one of the most important factors in building up popular support for the system. The state bureaucracy provided a unique historic chance for the poor and uneducated but imprudent i n dividuals and their families. Until recently, a basic commandment was quoted in the communist bloc as important to everybody wishing to make a career in the system: "Remember, never try to be too professional, never try to be too intelligent. That will ge t you killed." But now there is an obvious necessity to diminish the number of bureaucratic positions in order to save resources and time. There is also a need to promote professionals instead of political activists. The fate of bureaucracy - a spinal cord of the system - seems to be undefined. The differences between social stratification as a foundation of political systems in democratic, underdeveloped, and communist countries are illustrated by Diagram 1. Achieving Internal Equilibrium. Despite the curi o sity of social stratification under communist regimes, there are quite logical rules hidden behind this structure, stabilizing the whole system. The system's balance depends on the availability of economic recources. The centrally planned economy and its s tate-owned companies are a source of state spending for different purposes. A certain balance of spending is essential for achieving an internal socioeconomic equilibrium. At least a half of all resources goes to the state budget. The rest of the gross na t ional product is strictly controlled by central planners. Thus, consumption is not a result of economic development but is dictated by the central plan. The level of consumption and average salary do not reflect the economy's ability to produce consumer g o ods, but reflects the planner's conviction that salaries offered to employees, combined with expenditures for repressive forces, are sufficient to maintain the social order. The costs of the social order depend mostly on the extent of resistance. Tbat's w h y the situation in Poland differs from that of Rumania, for example. The cheaper the social order, the more resources that can be diverted from consumption to state projects, which are the main objects of the state's care because they represent a "state p ower" factor.
classd emocratic societu
upper classi k
underdeveloped societup ower class
communist societ%j D iagnam I Social stratification and ipo 1 it i ca 1 systemsState investment projects CD CD C) 0 C) CD C) Cl State budget
S 0 t ECONOMY c a i t a ee M s p C R e 0 e C n U P r y r IL e U t e y 8 M b P e t n e i 0 f 0 n i- 'n IL S 0 C I E T Y
Diagnam 2
.Socioeconomic equilibrium in communist society.17here are additional factors that influence the socioeconom ic equilibrium of a communist country. One of them is state employee benefits that are directly connected with the social prestige of the bureaucracy. The more privileges that are available for the bureaucracy, the stronger is bureaucratic careerism, whic h solidifies the whole balance of the system. On the other hand, the vast majority of the society depends on some subsidies pouring from the state budget. From our point of view, the most important part of this 99social security" factor are subsidies in ar e as of housing, food prices, health care, and education. Since employees of the socialized sector are beneficiaries of this factor, changes of expenditures for these purposes influence the whole system's equilibrium. Diagram 2 shows the main factors influe ncing the socioeconomic balance of the communist society and the position of a centrally planned economy as the source of funds for maintaining social order and supporting the state power factor, which is the ultimate goal of the whole system. CENTRALLY PLANNED ECONONIY This has been the core of the communist system from its very beginning. Remarks from such prominent economists as Ludwig von Mises and Friedrich Hayek that such a system would ultimately be inefficient were neglected. At the early stage of communism, state central planning seemed to be triumphant and, to some degree, was backed with enthusiasm by many Western economists who warned that the West would be surpassed economically by Soviet-style nations. Early successes of communist economies, h owever, were only short-terni, in that they relied on a concentration of all available resources in order to obtain a high rate of economic growth. T'his effect could last only as long as the pre-communist market price structure was maintained. But, since communist states intentionally destroyed market forces and granted unto themselves the right of price regulations, their economies became blind. Price Restructuring, Now the greatest plague of centrally planned economies is a lack of sense of locality. Si n ce state-controlled prices ceased to be controlled, inflation has shot up. Central planning jerks convulsively from one direction to another. Much investment is wasted. Pollution is the most intense in the world, and in the system that touts social justic e as the most important issue, there is more inequality than ever. That is why governments of communist nations follow so willingly the World Bank's advice to begin reforms by flrst implementing price reforms. Rulers think that this way they can revive the market price structure and restore the system's orientation in economic surroundings. But it appears to be not so easy. A free market is not simply an implementation of a price equilibrium. A price equilibrium is not even a beginning of market revival, no r should it be taken seriously as a prerequisite for major reforms. Look at the example of Yugoslavia. Yugoslavs implemented price equilibrium in the late 1960s. So what happened? The economic crisis seems to be as painful as in Poland where the price dise q uilibrium has been intentionally developed for many years. Tlat is because it is impossible to build up a free market price structure on the surface of fully centrally planned economic and social structure. Tlie resistance to price restructuring does not c ome merely from those who don't like it. Nobody likes price hikes. Tle real resistance comes from the communist system's substance, from all elements of the economy and social stratification. People resist price hikes mainly because they doubt that they c a n bring any positive result, because no link between price changes and productivity exists in a communist system. But this is an element the people have to live with; they are a part of this element, too. Thus, communist countries should not start free ma rket-oriented reforms with price reform but with introducing a new legal 6
system, a new civil code that will be able to secure property Tights and accelerate the emergence of a capital market. 11eTe is a substantial difference between the meaning of th e term "market" in the two systems. In a free market system the market is a complex structure regulating the behavior of three market "actors": consumers, entrepreneurs, and government agencies. The government plays a specific moderator's role in a free m a rket system. In the centrally planned economy, the market means something entirely different: it is only a small margin of the economy where consumers can buy some goods with their salaries. Only this part of the economy is called market. Thus, one actor - the consumer - is being kept on a short leash by central planners. Another one - the entrepreneur - simply does not exist. The only independent and really decisive actor remains the state which is an amalgamation of the communist party structure, the gov e rnment and its agencies, and the socialized sector Of the economy. The difference between a free market complex and the market in a centrally planned economy is shown in Diagram 3 and 4. Thus, the national economy in a centrally planned system is divided i nto two areas which are not connected directly: the market area and non-market area. The primary part of the economy is the non-maTket one. The market area is of secondary importance and represents the consumption factor from Diagram 2. The GNP is distrib u ted in two sequences: the extent of consumption is not decided before the level of investment is stabilized. The level of consumption is what remains after the investment plan is executed. Interrelations between market and non-maTket areas are shown by Di agram 5.
The system of nomenklatura has stopped working. It has been frequently misunderstood in the West. This system is very unusual because it gives more power to informal (non-noTmative) institutional structures than to formal (normative) ones. That lets power elites implement the most deMOCTatic-looking legal systems in the most oppressive regimes in the world. Ibis allowed, for instance, Stalin to celebrate the abandonment of the death penalty in the Soviet penal code while millions of people were dying in the Gulag. The nomenklatUTa system is not reflected b y law. The only legal basis for communist party power is its role as a "vanguard" as mentioned in the communist state's constitution. The real basis of its power, however, is the social contract formed and solidified by years of Stalinist repressions. The p osition of the Communist Party hierarchy in the state's structure is shown by Diagram 6. No One to Blame. Now the nomenklatura system has stopped working. Poland is a forerunner of the nomenklatura's decay, and this can explain the phenomenon of the Polis h case. Formally and legally, the Polish Communist Party had no power. It had always relied upon vocal orders, never written decisions, to carry out its wishes. That is why all the documents found by Solidarity activists investigating illegal practices con t ained only signatures of people from administrative institutions - Communist Party leaders appeared to be free of any charges. Even Mr. Gierek, Polish ruler and the first secretary of the powerful Communist Party Central Committee, pretended be was not in a position to make any important political or economic decisions. He was fired from his post but since there was no legal evidence against him, he was never punished. After martial law was implemented in Poland, the Polish Sejm became one of the busiest p arliaments of the7
I.. Market complex in a -free-market economij.F CT M Comsumer goods & services 0 a investment goods & services v r r e e Labor market r k n m e Capital market e n t ID, Entrepreneurs
Diagram 3 Market complex in a free-market and Soviet-type economies.2 . 411arket" in a -central 19-planned economg o n s u m e r S@ Consumer goods & services Ab market 1w .... ..............X W. -K AW nu, 0. W 40.'*"*. W. .-W OW 4W APAW"PAPAW low, w V. Alp, S t a t e *-**@ AWI& AOI mon PO mr, Or-4 li '-t ... . . Ap 'A@ Aw "Att _.Aw NIA;P@ W-W ...&. 10,0. a r e a X 'WAwAwArAwArAr'w ' ' I I @F. K@? @- @ 17- & W. C r. @sC T M 0 [@o v e r
81. Market complex in a +Pee-manket economu. C o n s u m. e r s, F Comsumen goods & services CT 0 1 0 a Investment goods set-vices V r r e e . Labor market r k n M e Capital market e n t Entrepreneurs
Diagram 4 Market complex in a free-market and Yugoslavian-t-ype economies,2 . Market in Yugoslav ian-tUpe economU. Con s um F Consumer goods services 0 market m Investment goods ser-v i c es r a market e r ik Labor market 9 e n t ate monopoll%ij s 44 a r e a ----------- C 0
9D agnam 5 "Market" and non-market areas in a centrally- planned economy Mar.ket area o n - m a r ]k e a r e a wages Consumers State budget --- Taxes t .[subs i - a x d e S ies s el Demand St State comp projects
production delivenuS pp1w Consumer Investment deliverg goods goods
The "nomenklaturall and an organization of a communist state.
EXECLYrIVE AREA POWER AREA EXECLYrIVE AREAC entna I Par-l iament plan ...... Communist PantluLl Central Committee Branch M i n i str-W Government
Regional T r u s t Committee 4p, W Regional dm i n istrat ion
CompanW trict Committee District Idministraticown&villag Comparaw's Inm a_ L a nd division divi ion Town illage committees dm i n i st na t i o T %t I Jon Co n gc
P' an = r Ban Mini st1 0
world, issuing hundreds of new regulations to prevent blaming the administration for further Communist Party apparatus power abuses. The Commmunist Party has never recovered from this blow, and its informal power weakened as never before. It was also a he a vy blow to the nomen1datura system, and it explains why Poland under military rule turned out to be one of the most liberal countries in Eastern Europe. Back to the Future. life has become uneasy in the East not only for the ordinary people but also for t h eir rulers. There are many questions coming out of the communist Pandora's box which, if not answered properly, may result in-hopeIessness and violence. That is why the governments and power elites desperately seek for any pragmatic answer to the current c risis, no matter how unpleasant. Now, the ultimate answer exists, which I am going to offer during my second lecture. It is not an easy one to accept, but people facing a firing squad are usually well-prepared to make many concessions, no matter how great . Tle answer lies in the popular Polish joke, that "socialism is the longest way from capitalism to capitalism." Some communist governments understand the problem better than others. The Parliament in Budapest, Hungary, has just approved a Law on Corporate Association which, from the beginning of next year, is intended to let the private sector blossom, liberate the movement of capital, and allow Western companies to buy Hungarian ones. Hungarians now seem to be taking capitalism out of the closet and on to the statute books. The communist nations, after decades of economic and social experiments, now find themselves trapped in a time machine, which is taking them back to the reality they had passed. They have to witness history again, but this time in rever s e: from a centrally planned economy to a free market, from the flat and equalized society to social and political diversification, from nomenklatura to democracy, but also - from hopelessness to newborn hope. It is a long, rocky, and dangerous path, but w hat the West can do is help them mount the mule.}}


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I recently got some FCS JF-1 fins and rode them a couple times and they weren't exactly what i was looking for. They're a little too drivey for me and work better in good hollow surf rather than average surf which is what i was wanting. The fins are still in spankin' new shape. Looking for something else. FCS fin systems only. Let me know what you guys have.
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Dec 12, 2013, 12:56 AM #1
Looking for a trade for my brand new FCS JF-1 fins
FCS TC Aqualines...
Dec 12, 2013, 01:57 AM #3


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Sex beast jailed for life for raping grieving mum beside her six-year-old's son grave
A BRUTAL sex attacker has been jailed for life after he twice raped a grieving mum as she placed flowers on the grave of her six-year-old son.
Christopher Rode, 30, knocked his 56-year-old victim unconscious with a small brass and ceramic statue he snatched from a gravestone.
A court heard how the vile sex beast twice raped the mourning mum, whom he did not know, on the 20th anniversary of her son's death.
Rode muttered the chilling words "I will kill you" as he forced his victim to the ground and covered her mouth with his hand to stop her from screaming in Slough Cemetery and Crematorium in Stoke Road, Slough, Berks, on May 9.
Twisted Rode was sentenced to life in prison after he admitted assault causing grievous bodily harm and two counts of rape.
Sentencing at Reading Crown Court, Judge John Reddihough said Rode will serve at least 14 years.
"The horrendous attack on this woman while she was paying her respects to a loved one in a public crematorium was a truly dreadful crime which shocked the local community"Detective Inspector Stuart Brangwin, of Berkshire Police
The court heard notorious criminal Rode, from Slough, struck from the bushes when the mum was about to walk back to her car.
Michael Roques, prosecuting, said: "She heard a rustling sound coming from a nearby hedge and turned to look.
"The individual struck her four times to the face using some sort of weapon.
"She described regaining consciousness lying on her front, pinned between the car itself and the kerb and bleeding from her face onto the roadway.
"She attempted to scream for help but a gloved hand was covering her mouth.
"She tried to clamp her legs together in order to prevent her lower garments being further removed."
A man has been sentenced to life, minimum 14yrs 9mnths,for rape and GBH at a Slough cemetery https://t.co/rJIfyVZjpj pic.twitter.com/B1cgRo681e— TVP Slough (@TVP_Slough) July 11, 2016
Mr Roques added: "She described struggling over the course of the attack and telling her attacker that it was the anniversary of her son's death and she was tidying her son's grave.
"This resulted in the defendant leaning very close to her ear and saying 'I will kill you'. She described his voice as very, very angry.
"After some minutes she managed to get herself into her car. In the footwell of the driver door was a ceramic and brass bear statue. This it seems was the weapon with which she had been struck, it having been removed from part of a grave decoration within the cemetery."
The court heard sick Rode has numerous convictions, including beating a pregnant ex.
Speaking after sentencing, investigating officer, Detective Inspector Stuart Brangwin, from Berkshire Police, said: "The horrendous attack on this woman while she was paying her respects to a loved one in a public crematorium was a truly dreadful crime which shocked the local community."


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Start your own Yelp like website! Meeplace is a Review & Rating Script, like a yelp clone, that you can download, install and use to create your own review site. Meeplace provides you with a easy solution software that allows your visitors to submit their own reviews on businesses, cars, movies or whatever content you have listed on your site. Meeplace also is a great way for your visitors to talk about their own personal experiences with companies, car dealerships or even the local handyman. Use Meeplace to start your own business review site, product review site or directory site in minutes. Also feel secure knowing our code is not outsourced. All our code is created internally in our company. Now with: - Facebook connect - Multi-Language - Custom Theme Changer - Multiple Admins and Moderators - Featured Listings - Photo Galleries - Claim listings - Auto Email Notifications - Newsletter system - CMS for custom pages - PHP & MysqlVisit publisher site: Meeplace
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Bottom Line: No, I would not recommend this to a friend


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After his declaration, Doug is desperate to clear the air - but Ste is distracted by the arrival of his new man Adam. While Ste makes his choice, Doug has a change of heart.
Meanwhile, Maddie cements her social status by hosting a gig at the coffee shop. However, when a guilty Esther realises how much pain she's caused Tilly, she decides to do something drastic to stop the rumours.
Elsewhere, George tries to coax Phoebe into making new friends.
Also today, Cindy's birthday meal for Tony proves to be even more of a surprise than she expected.
Writer: Laura McCann
Director: Tracey Rooney
> Hollyoaks star PJ Brennan chats to Digital Spy about the Doug and Ste storyline