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Recent results suggest a paradigm shift from viewing inorganic phosphate as a passive requirement for basic cell functions to an active regulator of cell behavior. We have previously demonstrated that elevated concentrations of phosphate increased cell proliferation and expression of pro-tumorigenic genes such as Fra-1 and osteopontin in a pre-osteoblast cell line. Therefore, we hypothesized that elevated phosphate concentrations would promote cell transformation in vitro and tumorigenesis in vivo. Supplementation of medium with phosphate increased anchorage independent transformation and proliferation of Balb/C mouse JB6 epidermal cells, activation of N-ras, ERK1/2, and AP-1, and increased gene expression of Fra-1, COX-2, and osteopontin in a dose-dependent manner. These in vitro results led to the hypothesis that varying the levels of dietary inorganic phosphate would alter tumorigenesis in the mouse model of skin carcinogenesis. Female FVB/N mice were treated with 7,12-dimethylbenz[a]anthracene/12-O-tetradecanolyphorbol-13-acetate (DMBA/TPA) and fed high or low phosphate diets (1.2 versus 0.2% of the diet) for 19 weeks. The high phosphate diet increased skin papilloma number by approximately 50% without changing feed intake and body weights. High dietary phosphate increased serum concentrations of phosphate, parathyroid hormone, and osteopontin and decreased serum concentrations of calcium. Thus, we conclude that elevated phosphate promotes cell transformation and skin tumorigenesis partly by increasing the availability of phosphate for activation of N-ras and its downstream targets, which defines dietary phosphate as a novel target for chemoprevention.
Tumorigenesis is a multistage, complex process often involving both genetic and environmental or lifestyle factors. Lifestyle factors, such as diet, can have a profound influence on the initiation, progression, and/or recovery from disease. This idea is supported by studies evaluating the effects of human migration on the incidence of particular diseases including cancer [reviewed in (1)]. A general estimate of the potential contribution of diet to cancer ranges from 10-70% (2). Diet represents an environmental factor that can be relatively easily manipulated and it is becoming increasingly apparent that diet can have profound effects on functional genomics (3). However, sufficient information does not currently exist to permit a comprehensive utilization of individual dietary components in the prevention, intervention, and treatment of disease. A number of in vitro studies have suggested that inorganic phosphate (Pi) and phosphate transport are necessary for cell growth, essentially acting as a mitogen (4-7), however little research has been directed at determining the mechanism and the consequences of elevated Pi in vivo. Our previous results in a pre-osteoblast cell line suggested that, in vitro, elevated Pi led to increased cell proliferation (8) as well as activation of ERK1/2, changes in protein and gene expression of AP-1 proteins, and increased expression of transformation associated proteins such as osteopontin (OPN), Fra-1, and cyclin D1 (8-12). In addition, elevated Pi promotes Akt-ERK1/2-Mnk1 signaling, cap-dependent protein translation, and growth in human lung cells (13). Taken together the data suggest that the elevated levels of available Pi may be an important driving factor to the growth and transformation potential of cells.
The transformation sensitive epidermal cell line JB6 is recognized as an excellent model to study multi-stage tumor promotion including the effects of bioactive food components such as Pi on the process of transformation (14). When treated with various tumor promoters these cells respond with anchorage independent growth in soft agar and tumorigenicity (15). Many key factors necessary for various steps of the transformation process have been identified and include: activation of members of the AP-1 transcription factor family, an increase in cyclins A, B1, and D1, activation of signaling proteins ERK1/2 and iNOS and increased expression of the extracellular matrix protein osteopontin [reviewed in (16)]. Members of the AP-1 transcription factor family (17) have also been identified as attractive targets in chemoprevention because their activation is linked to proliferation, transformation, and inflammation [reviewed in (18)] in a number of cell types and tissues including skin (19). A number of food factors have been demonstrated to reduce AP-1 activity and thereby reduce the associated transformation properties [reviewed in (18, 20)]. A gene/protein that is also tightly associated with transformation, inflammation, and metastasis is OPN, an extracellular matrix and circulating factor (21-23). Osteopontin influences cell function by acting as a cytokine through its ability to bind integrin receptors. Recently, OPN was found to be regulated by AP-1 under transforming conditions in the JB6 transformation model (24) and to be strongly regulated by Pi (10). The cellular and molecular events required for transformation in this model have been validated in vivo using the 7,12-dimethylbenz[a]anthracene/12-O-tetradecanolyphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis mouse model (23), which is commonly used to study the initiation, promotion, and progression of carcinogenesis (25). The defined molecular mechanisms make the JB6 and two-stage skin carcinogenesis models an excellent system to study the effect of Pi on cell transformation and tumorigenesis.
The ras family (H, K, N, R) of small GTP binding proteins (approximately 21 kDa) functions as important signaling proteins in controlling proliferation, transformation, and tumor invasion [reviewed in (26)]. Ras proteins are activated in the GTP bound state and are known to be central to many events related to proliferation, transformation, and tumor invasion through downstream effectors ERK1/2 and Akt. Ras regulated proliferation and transformation is linked to the regulation of proteins such as cyclin D1, the AP-1 transcription factor Fra-1, and osteopontin all of which we have previously demonstrated as responsive to elevated Pi (8, 10, 11). Investigations into the role of ras and tumorigenesis have focused on activating mutations, however, overexpression of ras without activating mutations is also capable of transforming cells (27, 28). Although in vivo papilloma formation is known to involve activating mutations in the H-ras isoform and activation of AP-1 transcription (29), the H-ras null mice do form papillomas with 62% having k-ras mutations and 38% having no ras mutations (30).
The objective of the current study was to test the hypothesis that elevated levels of Pi promote proliferation and anchorage independent growth of transformation sensitive epidermal cell line JB6 and skin tumorigenesis in DMBA/TPA treated female FVB/N mice. Furthermore, we explored the molecular mechanism by which Pi alters tumorigenesis and hypothesized that Pi promotes activity of ras, ERK1/2, and AP-1 and ultimately gene expression of Fra-1, COX-2, and osteopontin in a dose-dependent manner.
Transformation-sensitive JB6 (clone 41) mouse epidermal cells were cultured in monolayers at 37 °C and 5% CO2 using Eagle's minimal essential medium (EMEM; 1 mM Pi; Invitrogen, Carlsbad, CA) containing 4% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA) supplemented with 2 mM l-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). GDPβS was purchased from Calbiochem (San Diego, CA). Unless noted all experiments were performed in EMEM which contains 1mM Pi and concentrations listed in the figures are final Pi medium concentrations. Added Pi is in the form of NaPO4, pH 7.4 (Sigma).
Cell viability was measured using XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} assay according to manufacturer's protocol (Promega, Madison, WI). JB6 cells were plated at 5×103 cells /100 μL per well in 96 well plates. After 24 hr, plated cells were treated with 1 and 2mM Pi (1, 2, and 3mM final) for 36 hr. The change in absorbance was measured 1 hr after addition of XTT assay reagent on a BioRad Lumimark plate reader (BioRad Laboratories, Hercules, CA). The results are from 6 replicates/treatment. Cell counts were performed generally as described previously (8). JB6 cells were plated at 5×104 cells/well of a 12 well plate and cell counts were performed after 96 hours in medium containing 1 or 3 mM Pi (final). Cell cycle analysis was performed by Flow Cytometry. JB6 cells were subcultured on 10cM plates and after 48 hours, 2mM phosphate was added, or not, for a final concentration of 1 and 3mM. After 36 hours cells were lifted with Accutase (Innovative Cell Technologies Inc., San Diego, CA), washed with PBS, fixed in ethanol, and washed twice. Cells were stained with propidium iodide (Roche) at 0.1mg/ml final with RNase A (Invitrogen) 1-hour prior to analysis by flow cytometry (Accuri flow cytometer, Ann Arbor, MI).
Promotion of neoplastic transformation assays were performed as described previously (15). In a 60-mm tissue culture dish JB6 cells (1×104) were resuspended in 1.5 mL of 0.33% agar in EMEM (1 mM Pi) with 10% FBS and layered over 7 mL of 0.5% agar in EMEM with 10% FBS. Both layers of agar were supplemented with Pi (1.2, 2.2, or 3.7 mM NaPO4) in the presence of DMSO or 10 ng/mL phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; Alexis, San Diego, CA) in DMSO. The cells were cultured at 36 °C for 14 d, stained with neutral red (Sigma) diluted 1:75 in PBS. Colonies were counted by an automated image analysis system supported by Image Pro-Plus (version 3.0.1) software (Media Cybernetics, Silver Spring, MD). Colonies of greater than 8 cells were scored. The transformation responses are presented as average number of colonies formed per 60-mm tissue culture dish (5 replicates/treatment).
JB6 cells were cultured as above and nuclear isolation and immunoblotting performed as described previously (8). Thirty μg of lysate was resolved on a 10% SDS-polyacrylamide gel. All antibodies were purchased from (Santa Cruz Biotechnologies Inc., Santa Cruz, CA), except ERK1/2 from (Promega, Madison, WI).
Total cell RNA was prepared using Trizol Reagent (Invitrogen) according to manufacturer's recommendations. A total of 10 μg of RNA was loaded per lane and separated by electrophoresis through a 1% formaldehyde-agarose gel. The RNA was transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech, Inc., Piscataway, NJ), crosslinked by UV irradiation, and baked at 80 °C. The 32P-labelled probes were prepared using a random prime labeling kit (Roche Diagnostics Corp., Indianapolis, IN). Between successive probes, blots were stripped by treatment with boiling 0.1% SDS. Radiochemicals were obtained from Perkin Elmer Life Sciences Inc. (Boston, MA). The OPN, Fra-1, and actin probes used for Northern blotting have been described previously (8). The cDNA probe for Cox-2 (ptgs2) was created by RT-PCR using primers 5'-ATGCTCTTCCGAGCTGTGCT-3 ' AND 5 '-CAGCTCAGTTGAACGCCTTT-3' and Egr1 using primers (5'-ATGGCAGCGGCCCAAGGCCGAGATGCAATT-3') and (5'-GCAAATTTCAATTGTCCTGGG-3') and cloned into vector TOPO.2.1 (Invitrogen).
Transformation-sensitive JB6 cells were seeded at 2×104 per well in a 24 well plate. After 24 hr, plated cells were transfected with 4×AP-1 luciferase construct (500 ng/well) and Renilla (50ng/well) (Promega) using Fugene (Roche). After another 24 hr the medium was replaced with DMEM and 0.2% FBS (6 replicates/treatment). After another 24 hr, the medium was supplemented with 1, 2, or 3 mM Pi for 6 hrs and DMSO (control) or 10ng/mL TPA in DMSO as indicated. The cells were washed in PBS and harvested in 1Xlysis buffer. Firefly luciferase and Renilla activities were measured with a luciferase kit from Promega (Madison, WI) according to manufacturer's recommendations on a Turner Biosystems Inc. microplate luminometer (Sunnyvale CA).
Transformation-sensitive JB6 cells were serum starved overnight, supplemented with 2 mM NaPO4 for 0, 5, 10, 15, 30, and 60 min, and harvested in PBS. To separate nuclear and cytoplasmic fractions, cells were first lysed in a buffer of 0.25 mM HEPES, 50 mM KCl, 2 mM PMSF, 100 μM DTT, 0.5% NP-40, and protease inhibitors (at a final concentration of 2.0 μg/mL aprotinin, 2.0 μg/mL leupeptin, 1.0 μg/mL pepstatin, 100 μM sodium ortho vanadate, and 1 mM DTT). After centrifugation and removal of the cytoplasmic fraction, the nuclear pellet was lysed in an extraction buffer of 0.25 mM HEPES, 50 mM KCl, 2 mM PMSF, 100 μM DTT, 10% glycerol, and protease inhibitors at the concentrations indicated above. The protein concentration of cell lysates was determined with a BCA Protein Assay Reagent kit (Biorad). Radiolabeled consensus AP-1 oligonucleotides, purchased from Santa Cruz Biotechnology (Santa Cruz CA), were generated according to manufacturer's protocol (Promega). DNA binding reactions were performed using 5 μg of nuclear extract, 5X Gel Shift Binding Buffer (Promega), labeled oligonucleotide, and nuclease-free water. Reactions were separated on 6% DNA retardation gels (Invitrogen). After separation the gels were vacuum dried and exposed to film. The supershift assay was performed using nuclear lysate from a sample that was exposed for 5 min with 2 mM supplemented Pi. Labeled oligonucleotide and nuclear lysate were combined and incubated for 15 min followed by addition of antibodies of the specific AP-1 family members. All antibodies were purchased from Santa Cruz.
JB6 cells were serum starved overnight and supplemented with 0, 2, or 4 mM NaPO4 for 5 min (in addition to the 1 mM in the medium). Ras activity was measured using the Ras-Binding Domain of Raf-1 to pulldown active ras according to manufacturer's protocol (Cell BioLabs, San Diego, CA). Blots of whole cell lysate (50 μg) from the input were probed with antibodies to total ERK1/2 (Promega) and phospho-ERK1/2 (Santa Cruz). For time course experiments JB6 cells were supplemented with 2 mM Pi for 0, 2.5, 5, 10, or 15 min and ras activation assay performed. To investigate the requirement of phosphate transport 1 mM foscarnet (phosphonoformic acid) was added 1 hr prior to the supplementation of 2mM Pi. Following separation by SDS polyacrylamide gel electrophoresis, the resulting membrane was probed with a pan-ras antibody followed by N-ras, R-ras, H-ras, and K-ras specific antibodies (Pan-ras antibody was from Cell BioLabs, all isoform specific antibodies were purchased from Santa Cruz).
Mice (female FVB/N) were obtained at 4-5 wk of age from NCI-Frederick Animal Production Area, Frederick Cancer Research and Development Center (Frederick, MD). Mice were housed in a facility with controlled conditions (temperature: 21-24°C, humidity: 40-70%, and light/dark cycle: 12 hr light/12 hr dark). Until 8 wk of age, mice were fed ad libitum NIH-31 diet (1.1% phosphate; Harlan Teklad, Indianapolis, IN). At 8 wk of age, mice were randomly assigned to either a low phosphate diet (LPD; 0.2% phosphate) or a high phosphate diet (HPD; 1.2% phosphate) (Table 1) both manufactured by TestDiet (catalog # 58299 and # 25608, respectively, Purina Mills, Richmond, IN). For the two-stage skin carcinogenesis model, 14 mice/group were shaved at 8 wk of age. After 2 d, mice were topically treated with 400 nM of 7,12-dimethylbenz [a]anthracene (DMBA-Sigma) in 200 μL of acetone. Starting 14 d after DMBA initiation, the mice were topically treated with 10 nM of TPA (Alexis, San Diego, CA) in 200μL of acetone twice a week. Mice were visually examined weekly for skin papilloma and squamous cell carcinoma number and size. Every 3 wks individual body weights and food disappearance per cage (3 or 4 mice/cage) were measured. Mice were sacrificed at 27 wk of age.
For the serum analysis, 5 mice/group were fed starting at 8 wks of age the experimental diets for 5 wk. Serum samples were collected from the mice by cardiac puncture. Serum concentrations of phosphorus, calcium, magnesium, creatinine, uric acid, and total protein were measured by the Laboratory of Experimental Immunology (LEI), National Cancer Institute-Frederick, MD. Serum concentrations of parathyroid hormone (PTH) and insulin growth factor I (IGF-I) were measured by AniLytics (Gaithersburg, MD). In addition, 6 mice/group were fed starting at 8 wks of age diets containing 0.2 or 1.2% of Pi for 2 wk and then topically treated with 200 μL of acetone (control) or TPA (10 nM) in 200 μL of acetone. Serum samples were collected by cardiac puncture 6 hr after TPA treatment. Serum concentrations of OPN were measured by ELISA (R&D Systems). Animal care and experimental procedures were conducted with approval of the NCI-Frederick Animal Care and Use Committee.
Statistical analysis was conducted using SAS Version 9.1 (SAS, Inc., Cary, NC). For the cell culture studies a one-way ANOVA was used. When significant effects were detected (F-test significant at P < 0.05) we used a two-sided t test to compare each of the two higher Pi concentrations with the lowest Pi concentration. For the animal studies, papilloma size and mouse weight data were analyzed as a repeated measures study using the mixed models procedure (PROC MIXED). Papilloma size data were transformed to the log (X+0.5) scale so that the data were approximately normal distributed. The fixed effects in the model were dietary Pi (low, high), time (week of age), and the interaction of dietary phosphate and time. A completely unrestricted variance-covariance structure was used to account for repeated measures taken on individual mice across time. The effects of dietary Pi were evaluated by comparing the estimated values at each time point with each other using two-sided t tests in the LSMEANS statements. Papilloma and squamous cell carcinoma incidence data of the two dietary Pi groups were compared by using Fisher's Exact test in PROC FREQ. Concentrations of serum variables of the two dietary Pi groups were compared using a two-sided Student's t test. Significance was declared at P ≤ 0.05 and trends toward significance were declared at P ≤ 0.10. Unadjusted means and standard errors of means (SEM) are presented.
It is becoming increasingly apparent that diet can have profound effects on functional genomics and represents an area of research that has yet to be exploited for potential health benefits. Inorganic phosphate (Pi) is a common dietary component that may directly alter cell and tissue behavior in just such a manner. Almost four decades ago studies noted that contact inhibited 3T3 cells responded to serum stimulation with a rapid increase in Pi transport (31) and described Pi as a limiting nutrient in proliferation (4-6) capable of actively altering cell growth properties (32) and transformation (7). Recent in vitro results suggest that Pi is capable of stimulating specific signal transduction pathways including ERK1/2 and Akt (8, 12, 13) and suggest a paradigm shift from viewing inorganic phosphate as a passive requirement in these processes to an active regulator, thereby defining a novel mitogenic signal. Based on these in vitro studies we hypothesized that Pi is in fact a mitogen. It would then follow that the level of available Pi would actively alter the transformation potential of cells in response to a tumor promoting event.
We utilized a number of assays to determine if elevated Pi promotes proliferation. Throughout our in vitro studies we have used a spectrum of final Pi concentrations to cover the physiological range of both mice and humans. Elevated Pi promoted cell proliferation as measured by cell viability (Figure 1A), cell number (Figure 1B), and the percent of cells in the S and G2/M phases of the cell cycle (Figure 1C). Additionally, elevation of Pi in a physiological range increased conversion of cells to anchorage independent growth in transformation sensitive JB6 cells (Figure 1D). Anchorage independent growth in transformation sensitive JB6 cells represents a well defined marker of transformation in vitro and correlates specifically with tumorigenicity in vivo (33). An increase of as little as 1 mM Pi alone increased growth and anchorage independent growth in soft agar (Figures 1A, D). Supplementation of 1 mM Pi is in addition to the 1mM Pi in the medium and indicates that elevated Pi alone is sufficient to enhance cell transformation. Furthermore, elevated Pi acted synergistically with TPA, which is commonly used to promote cell transformation in JB6 cells, to increase growth in soft agar (Figure 1D) indicating that Pi renders JB6 cells more sensitive to TPA induced transformation. The soft agar response correlated with a dose-dependent increase in gene expression of OPN, Fra-1, and Cox-2 (Figure 1E), which are linked to transformation in the JB6 model (22, 27). The augmentation of the TPA response by Pi was also correlated with the expression of osteopontin (Figure 1F). In particular, OPN (spp1) represents a gene/protein that is tightly associated with transformation, inflammation, and metastasis and is an extracellular matrix and circulating factor (21, 22). The growth of JB6 cells in soft agar has been shown to be dependent on OPN expression (34) and recently OPN was found to be regulated by AP-1 under transforming conditions in JB6 cells model (24). The expression of OPN is often considered a late response and to determine if elevated Pi resulted in an upstream response cells were analyzed for gene expression at earlier time points. The results revealed that, in fact, elevated Pi alters the Early growth response-1 (Egr1) gene within 30 minutes of exposure (Figure 1G). Egr1 is considered a marker of cell growth and proliferation and we have previously described it as a Pi responsive protein (35). Fra-1 and Cox-2 begin to rise later in the response and OPN does not change at these relatively early time points. In summary, our results suggest that high available Pi promotes proliferation and transformation as well as the gene expression changes necessary for transformation in JB6 cells.
To elucidate the molecular mechanism by which high available Pi promotes cell proliferation, transformation, and changes in gene expression, we focused on AP-1 and ras activation. Our previous in vitro studies suggested that Pi is a molecule capable of regulating specific signal transduction pathways including ERK1/2 and Akt and subsequent gene expression leading to increased proliferation (8, 12, 13). The strong link between AP-1 transcription factor activation and JB6 transformation (14, 36-38) and the increased expression of the AP-1 transcription factor Fra-1 in response to Pi (Figure 1E) suggested that the AP-1 transcription factor family may be responsive to Pi. Parallel to the soft agar response (Figure 1D), elevated Pi alone was sufficient to promote AP-1 activity and augment TPA in promoting AP-1 activity (Figure 2A). The effect of elevated Pi on AP-1 DNA binding was rapid (within 5 min; Figure 2B) and supershifts suggest the presence of at least the AP-1 transcription factors Fra-2, JunB, and FosB at this early time point (Figure 2C). In agreement with the rapid stimulation of these AP-1 proteins, Fra-2 and JunB have been previously demonstrated to be regulated within 30 to 45 min in response to serum stimulation and suggests elevated Pi induces a common, early growth signaling response (39). Although, JunB has also been linked to senescence promoting activities (40), the strong induction of proliferation by Pi suggests a positive role in this case. It is possible that the dimerization partner may determine the positive or negative effect of JunB on proliferation. We performed a Western blot of nuclear lysates from JB6 cells to determine if high Pi results in changes in AP-1 protein levels or posttranslational modifications. Results suggest a change in the protein status of Fra-2 with a rapid shift to a slower migrating form in response to elevated Pi (Figure 2D). Other AP-1 proteins did not appear to be significantly altered at these time points. To confirm changes in gene expression under these conditions RNA was analyzed for Egr1 expression and demonstrated a strong increase within 20 minutes of elevated Pi exposure (Figure 2E). In summary, our results suggest that one potential mechanism by which high available Pi promotes cell proliferation and transformation in JB6 cells and the gene expression changes necessary for transformation is by promoting AP-1 DNA binding.
Transformation of JB6 cells is dependent on activation of the Ras-Raf-MEK pathway (41). Our previous studies combining proteomic and microarray analysis in the pre-osteoblast cell line predicted that the ras family was involved in Pi signaling (10). As hypothesized, elevated Pi promoted within 5 min ras activity and ERK1/2 phosphorylation in JB6 cells (Figure 3A). At this early time point, elevated Pi primarily activated N-ras (Figure 3B). Reprobing stripped blots with antibodies to H-, K-, and R-ras showed no detectable change in response to Pi (data not shown) suggesting elevated Pi selectively activates N-ras. Quantification of 3 experiments suggested a significant time dependent increase of N-ras activity in response to elevated Pi (Figure 3C). The Pi-induced increase in N-ras activity at this early time point can be inhibited by blocking phosphate transport into the cell (Figure 3D), as evaluated by pretreatment with 1 mM of the known phosphate transport inhibitor phosphonoformic acid, also known as foscarnet (42). This provides strong evidence that availability of Pi alters N-ras activity and identifies Pi as a novel regulator of ras activity. To determine if GTP signaling occurs upstream of ERK1/2 activation JB6 cells were serum starved and pretreated overnight with GDPβS, a stable, inactive analog of GDP, and treated with phosphate for 10 minutes. The resulting Western blot revealed that inhibition of GTP signaling blocked phosphorylation of ERK1/2, suggesting the GTP signaling event is upstream (Figure 3E).
The N-ras knockout mice are viable and grow normally (43), however, these mice are less responsive to carcinogen induced tumors than wild-type mice and over-expression of wild-type N-ras has been demonstrated to increase the incidence of lymphomas (44). To our knowledge the N-ras null mouse has not been tested in the two-stage model. Our data suggest that normal activated N-ras cooperates with activated H-ras to enhance tumor formation, progression, or both. A precedent for such a scenario has been suggested using the N-ras null mice. This study demonstrated the stimulation of Raf and Rhoa by N-ras and Akt and simultaneous activation of cdc42 by K-ras to cooperate in transformation (45). Another study using a colon cancer model demonstrated that expression of active K-Ras (G12D) altered proliferation while active N-ras (G12D) altered apoptosis, again suggesting unique, non-overlapping, and cooperative roles for the ras isoforms in transformation (46). In summary, our results suggest that another potential mechanism by which high available Pi promotes cell proliferation, transformation, and gene expression in JB6 cells is by activating a Ras signaling network.
The JB6 transformation model has been demonstrated to be predictive of results in the two-stage skin carcinogenesis model. Consistent with the results in the JB6 model, high dietary phosphate (HPD, 1.2%Pi) increased skin papilloma number by 50% compared to low dietary phosphate (LPD, 0.2% Pi) in DMBA/TPA treated female FVB/N mice (Figure 4A&B). Similarly, in a recent study of lung tumorigenesis in mice, high dietary Pi (1.0%) increased tumor number and size compared to 0.5% Pi (47), suggesting a general promoting effect of high dietary phosphate level on tumorigenesis. Although all mice developed at least one papilloma and papillomas of similar size at the end of trial, there was a trend toward delayed incidence and growth (Figures 4B&C). Speculatively, mice on the HPD develop papillomas earlier and more papillomas may progress to squamous cell carcinoma than the mice on the LPD (HPD: 3.9% or 5 of 127 papillomas versus LPD: 2.3% or 2 of 86 papillomas), although more data is needed for statistical validation of these observations. However, the fact that the papillomas were smaller in size with the LPD and taken with a recent study that demonstrated that HPD stimulated increased cell proliferation and lung tumorigenesis of K-ras active mice (47) suggests that the HPD does accelerate proliferation. Neither of the Pi diets impacted body weight (Figure 4D) or feed consumption (not shown), which is consistent with the results in the lung tumorigenesis model (47). In summary, our results suggest that high dietary Pi promotes skin tumorigenesis in DMBA/TPA treated female FVB/N mice.
Altered serum phosphate might not only influence cell and tissue behavior by cell autonomous effects but also paracrine and endocrine effects. Levels of serum phosphate in mice and humans have been demonstrated to be positively correlated to parathyroid hormone (PTH) levels (48, 49). Although the receptor for PTH has been identified in rodent keratinocytes (50), the presence in human keratinocytes is not clear (51) and little is known about potential effects on cell behavior. We found that HPD increased serum concentrations of phosphate and PTH and decreased concentrations of calcium and uric acid, whereas serum IGF-I, magnesium, creatinine, and total protein concentrations were not affected (Table 2; Figure 5A). This suggests a specific rather than a general effect of dietary Pi on circulating factors. Here we report an additional circulating serum factor that is Pi responsive, OPN (Figure 5B). Osteopontin is known as both a survival factor and as a mediator of macrophage infiltration to the tumor site (52). Therefore, elevated circulating OPN concentrations might promote tumor initiation and progression. Consistent with our results in JB6 cells (Figure 1), high dietary Pi increased synergistically with TPA serum concentrations of OPN (Figure 5B). In summary, our results suggest that an additional mechanism by which changes in serum Pi might promote tumorigenesis is by increasing the levels of circulating OPN.
Our in vitro studies were performed using a spectrum of Pi concentrations physiologically relevant to both humans and mice. In humans steady-state serum phosphate concentrations generally range from 0.70 to 1.55 mM although levels differ slightly with sex and age. However, serum levels in humans can vary by as much as 1.2 mM following a high phosphate meal and remain stably altered from diet alone (53-55). Although basal serum phosphate levels are different between mice and humans the percent change we achieved in our study is in line with what is achievable in humans. The diets used herein resulted in a change in serum phosphate from 2.17 mM (LPD) to 3.42 mM (HPD), a 37% difference (Table 2). The change is proportional to differences found in humans in response to altered Pi diets (53, 54, 56). Results presented here suggest that differences in dietary Pi can cause significant long-term differences in steady-state serum phosphate levels in agreement with other published long-term diet studies in both rodents (57, 58) and humans (48, 53, 56, 59, 60). Due in part to the increased consumption of processed foods, the amount of Pi in the American diet continues to rise above levels already considered high by the FDA (59). The current dietary recommended allowance for Pi is 700-800 mg/day and the tolerable upper intake limit is 4,000 mg/d for adults. It is important to note that the diets reported here have increased Pi without corresponding changes in calcium as would be the case with a diet high in dairy (59). A human dietary equivalent of the daily Pi consumption of our mice is 500 mg/d for the LPD and 1,800 mg/d for the HPD. More than 50% of young and middle aged men consume greater than 1,600 mg/d and these calculations largely reflect "natural" sources of Pi and are therefore likely an underestimate (61).
In conclusion, the in vitro and in vivo results reveal that elevated Pi promotes cell transformation and skin tumorigenesis and suggest at least two different means. First, elevated Pi may act in a cell autonomous manner as a mitogen and promote proliferation and transformation through activation of a signaling pathway consisting of ras and ERK1/2. Elevated Pi may promote AP-1 transcriptional activation early in the response and ultimately changes in gene expression such as OPN, a known autocrine, paracrine, and endocrine factor later in the response. Secondly, elevated Pi increases circulating concentrations of PTH and OPN, both of which can act as endocrine factors and promote tumorigenesis. The significance of OPN in papilloma formation in the two-stage model has been recently demonstrated using the OPN null mice in which the lack of osteopontin markedly suppressed papilloma development, possibly through the prevention of apoptosis (62). As the amount of Pi in the human diet, and in particular the western diet, continues to rise (59), it will be important to fully understand the influence of Pi on cell and tissue function and the relationship to tumorigenesis (63). Furthermore, these studies identify dietary Pi as a novel target for chemoprevention.
GRB.Jr. and CEC are supported in part by grants from NIH/NCI (CA136059), NIH/NIAMS (AR056090), and an Emory University Research Committee grant. This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health (MRY, GB, CMP and NHC).
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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Ari D. Norman Turtle Pillbox
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Fantastic Beasts Newt Scamander Cosplay Bundle (Jacket, Vest, Tie)
Merlin's beard!
- Dress just as snazzy as Newt from the movie
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When I carry out indirect SPI programming it seems to fail, but my Flash is loaded correctly. The error depends on the mode pin settings.
Master Serial - iMPACT reports Failed and FPGA, but when I change the mode pins to SPI mode FPGA boots correctly.
SPI mode - iMPACT reports Successful and FPGA boots.
JTAG - iMPACT reports Failed and FPGA, but when I change the mode pins to SPI mode FPGA boots correctly.
This problem occurs because after indirect SPI programming, iMPACT prompts the FPGA to configure from the SPI PROM. If the mode pins are not set to SPI it fails. iMPACT then checks the FPGA DONE signal and will report a failure if it is low. A more detailed error message will be added in 10.1 Service Pack 1.
Date | 05/22/2014 |
Status | Archive |
Type | General Article |


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With a property portfolio heavily invested in Florida and Michigan, the Altman Cos. has been on the frontlines of the recession and its impact on multifamily market fundamentals, particularly when it comes to new development and community acquisition. While opportunities that pencil out for Altman have been few and far between, the company remains upbeat on firing up its development engine and even venturing into new markets via acquisition.
Altman CFO Tim Peterson, a veteran of Keystone Property Trust and an integral player in the Post Properties 1993 IPO, checked in with Multifamily Executive senior editor Chris Wood this week to share his thoughts on the capital markets, acquisition versus replacement cost, and the future of the GSEs.
MFE: It's been a while since you took Post through its IPO. Is that process anything you'd consider doing again?
PETERSON: Well, I was at Post when we were a private company and went through the IPO and was there for five or six years after that until I was pulled into doing something a little more entrepreneurial. But it was a great experience for me. I was young, and we were coming out of a difficult economy. I liked to say I wasn't the CFO there; I was the OFO—the Only Financial Officer. That in itself created a vacuum that helped to advance my career and learn a lot of things in a pretty compact time. I have fond memories—now that the all-nighters and some of the other things are in the past.
MFE: What is your take on the current state of multifamily capital markets and how they are impacting the Altman Cos. corporate strategies?
PETERSON: Right now we're pursuing what I would call an opportunistic acquisition strategy. There is quite a bit of capital out there for that, probably a lot more capital than opportunity. To date, we have not found many opportunities that we think justify some of the risk, and via deal underwriting sometimes you end up knowing too much. For acquisitions, we are looking at using fund equity or accessing GSE debt or some kind of bridge financing to GSE debt. The opportunities to date have not been large in number, have been super competitive and priced where every one of your assumptions has to be at least 90 percent right. To date, we have not been willing to take on that risk.
MFE: Are you looking for opportunities in your existing markets in Florida, across the southeast, and in Michigan?
PETERSON: We've looked primarily in the southeast and the southwest: Texas, Georgia, the Carolinas, and Florida. So we've tried to cast our net a little bit wider [then our existing portfolio] because of the scarcity of opportunities. We have started firing up our development opportunities. There are some equity forces that are becoming re-interested in that. There are a small percentage of players saying we are ready, so bring us the opportunities, and there is a larger wave behind them that recognizes that development is no longer redlined. They are starting to take a look at things but might not yet be in full invest mode. But the idea that putting equity into multifamily development is no longer taboo.
MFE: What are your thoughts on the likely future of the GSEs and how they fit into the multifamily financing arena?
PETERSON: I went into apartment finance out of business school because I looked at all of the other career and industry options and figured that this was far and away the simplest. My ability to assess geo-political risk is pretty limited, however. Obviously, the GSEs have a precarious financial position, but more importantly, they seem to also be facing a fair amount of political risk regarding what happens to them moving forward. That's impossible for me to handicap. If I step back and just try to be totally rational, the multifamily lending area is one where the GSEs have consistently been profitable in. Even today, while delinquencies have picked up, you'd hope that that profitability is continuing, and that rational thought prevails, and they continue to be lenders in this market. I don't think we can underestimate the amount of stability that the GSEs have brought to multifamily real estate's ability to ride through this economic storm. Compared to our brethren in the other food groups, we are certainly a lot stronger. You'd hope that a decision would be made to continue multifamily lending, but there's certainly political risk.
MFE: Do you see any bounce back from the banks, particularly when it comes to construction lending?
PETERSON: We've seen the banks beginning to recover. Obviously, Wells Fargo has put renewed focus on its permanent loan program, which had not been in the forefront for some time. We are seeing that in some of the other banks as well—DNC has a similar program. On the construction front, the banks are starting to look at lending, but frankly I think the equity is a little bit ahead of them. They want to do the business, they need to do the businesses to get back to sustainable profitability, but they are a little bit bipolar. They look at their financial statements and recognize the need to do construction lending, and then they get pounded by the regulators on their loan portfolio, so they get extremely nervous about adding anything to it. You have to catch them coming out of the right meeting. The banks are looking at it, but they are very focused on structure and sponsor. That should sort itself out into the beginning of a return to the lending market in the second half of next year. That's certainly my hope.


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The multiethnic metropolis
London, always a cosmopolitan city, grew steadily more polyglot and multicultural. The Commonwealth connection accounted for only part of the transformation. Despite restrictive immigration laws, the flux of refugees and asylum-seekers from many countries continued, and new communities of Vietnamese, Kurds, Somalis, Eritreans, Iraqis, Iranians, Brazilians, and Colombians sprang into being. Many of the foreigners settled into housing estates in the poorer parts of Inner London, particularly the crescent of inner boroughs to the east of the City. At the other end of the economic spectrum, London's position at the crossroads of the global economy brought transient ... (100 of 17,923 words)


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Tuesday, October 28, 2003
Yes on 14-15
I am a director of the Board of Crystal Springs Water District. I am also a Mt. Hood Meadows skier. While these two positions may seem contradictory, they do not need to be.
I doubt that there are many members of the RTRG group who have spent as much money and time at Meadows in the past decade as I have. I also believe that Mt. Hood Meadows plays an important role in the Hood River County economy.
That said, I support Measure 14-15. The north side of Mt. Hood, and the Crystal Springs watershed in particular, need the strongest protection available at this moment. To me, it is irrelevant who the prospective developer is. I don't care whether it is Meadows, William Smith Properties, or even someone local. You cannot develop in a watershed without putting it at risk of contamination.
The charter of the Crystal Springs Water District mandates that it must avoid the political arena; therefore I speak here on my own behalf. I beseech all of you voters in the county who, like myself, enjoy skiing at Meadows, to vote for Measure 14-15. There is nothing hypocritical about skiing in Meadows current location and choosing to protect our drinking water. Mt. Hood Meadows is a profitable and well-financed enterprise — it will remain so without having to expand on the north side of the mountain. Please do the right thing and vote — it's your water.
Gorham Blaine
What a footprint
I took Marvin Turner's suggestion to drive up Highway 35 to look at the size of the Cardinal Glass factory. What a wonderful facility and addition to our community. I am impressed by the way the land use laws were observed and followed in establishing this "family wage" company.
It is amazing to me to think that the size of the Cardinal Glass structure is the same size as the proposed Super Wal-Mart. This isn't even including the 12 acres of parking that will be necessary for this controversial footprint! I certainly hope that the county Planning Commissioners think long and hard about what this footprint will do to change our community and the character of our area.
If you haven't driven up Highway 35 I urge you to do so. I am sure you will also be amazed to think that this gargantuan Super Wal-Mart structure may be greeting you as you try to drive into our town. My, oh, my what a footprint! County Planning Commissioners, why is this even being considered?
Linda Drew
Hood River
No on 14-16
City residents, please consider the following as you mark your ballot on the waterfront initiative.
The waterfront property is currently zoned light industrial. The Port is requesting a zone change to permit mixed use which will include public parks. The Port wants park space. The Port also wants other uses of this valuable property. It is very possible to have both. The waterfront property is not God given. It is land that was developed by the Port of Hood River. Doesn't it stand to reason that if they developed this land they would care about its future? These people care very much about our community. They are not going to destroy the waterfront that they created.
Many of your friends are being disenfranchised by this proposed initiative. The waterfront belongs to ALL of the people living in the Port of Hood River district not only the people within the city limits of Hood River. I have lived in the Port of Hood River district for nearly 50 years and am not able to vote on this issue. This initiative is not fair. It is probably not legal.
Please don't cast your vote based on some emotional ploy like "Save our waterfront." Think as you mark your ballot.
Virginia Hosford
Hood River
Bridge too far
In early summer, the Lake Banch Road was closed so that the bridge across Divers Creek could be replaced. The end of September, the Lake Branch Road still had a barrier across it with a "Road Closed" sign on the barricade. It seemed absurd for it to take an entire summer to replace that bridge.
On Sept. 27, curiousity got the better of me and I decided to walk the three miles to the Divers Creek bridge to see how far along they were toward completion. When I arrived at Divers Creek, I found a brand new concrete bridge that replaced the old wooden structure. As I approached the bridge a car came up behind me. I flagged them down. I asked how they got past the barrier. The woman told me she had made local inquiry as to how to get to Wahtum Lake. She told me that the person she had talked to told her that she could drive around the barrier since the bridge had been completed.
When I see a barrier on a road with a "Road Closed" sign on it, I believe I'm breaking the law if I were to go around the barrier. As a result, I had to drive to the Lost Lake Campground entrance and go down the Lake Branch Road to get to Wahtum Lake. That means an 80-mile drive instead of the usual 54 miles (round trip) to get to my favorite lake.
In essence, the person who gave that woman permission to ignore the "Road Closed" was telling her it was okay to break the law.
If the bridge has been replaced, why don't they take down the barrier? Is that too much to ask?
Melvin Kenworthy
Hood River
No on 14-15
I wanted to take a moment of time to address the current Measure 14-15 which we will be voting on very soon.
I attended one of the public meetings Mt. Hood Meadows held this summer regarding the expansion of Cooper Spur. I was pleased that the company was reaching out to the public prior to submitting an application to Hood River County or the Forest Service for expansion. It was apparent to me that Meadows was very interested in being forthright about its plans and doing the right thing for the community, its employees, and its owners.
In the 37 years of Mt. Hood Meadows existence there have been numerous projects that have been proposed such as lifts at the ski area, and all of the proposals have been fought by the Hood River Valley Residents Committee. It is very apparent by the HRVRC's track record, that it was formed solely to combat anything Meadows proposes. I for one do not mind a system for checks and balances, however I do feel that there is such a thing as carrying a grudge too far, when checks and balances become obstacles designed to delay, at the expense of the taxpayers of Hood River County.
Mt. Hood Meadows in its history has not only grown to provide more and more jobs within our jobless county, but has continually striven to improve its efforts to include the county, and its residents in its decision making. Other businesses now benefit from this inclusionary thinking and the winter business Meadows creates for Hood River.
In addition, I've been up to Mt. Hood Meadows the last few summers. The area looks great, the employees and management are doing a good job as stewards of the land. I've been around Cooper Spur this fall, the area looks better than I have seen it in years. They have a beautiful base area all landscaped and have totally upgraded the buildings and have a brand new lift. Even the new signage is very rustic and attractive. I've even taken the opportunity to dine at the restaurant, a great experience.
Now Meadows wants to create a small quality expansion at Cooper Spur, outside of the Crystal Springs watershed. They wish to create more year-round jobs and continue to partner with Hood River businesses and promote local agriculture products to customers and suppliers of Mt. Hood Meadows and Cooper Spur as they have done in the past.
I believe that in the long run, Meadows will achieve its goal to expand Cooper Spur, and that's perfectly acceptable to me.
Measure 14-15 is nothing but another attempt to delay Mt. Hood Meadows' plans to expand Cooper Spur.
I'm voting no on Measure 14-15. I'm comfortable letting the Hood River County Planning Commission and the Board of Commissioners evaluate Meadows' proposal to expand Cooper Spur.
Eric Martinusen
Hood River
Yes on 14-15
Protect your water from special interests.
Opponents of Ballot Measure 14-15 have called supporters of the measure "special interests." That turns reality on its head. Public interest groups working to protect domestic drinking watershed are working to protect the public interest.
On the other hand, a good example of a special interest is a real estate developer planning to make big bucks from exploiting both public and private land in a very sensitive area of our forest zone.
That type of special interest was evident in the land trade that Mt. Hood Meadows made to Hood River County. Exchanging 640 acres of county forestland, most of it in the Crystal Springs watershed protection area, to a development corporation planning a resort is notoriously poor public policy. This land trade was protested at the hearings on Aug. 6 and 20, 2001, by citizens and by representatives of Crystal Springs and was opposed by the County Commissioner representing the part of the Valley most involved. But those very real concerns were ignored.
Apparently Mt. Hood Meadows and the county had been working together on the resort idea for some time before the hearing. The public testified but was not heard.
Ballot Measure 14-15 would protect against such exploitation by setting up a public check and opportunity for public input to protect the forestlands and domestic watersheds from unsound development.
Kate McCarthy
Yes on 14-15
I thought it was almost funny to read the recent letter that Measure 14-15 was the work of some elitist special interests. When has protecting the water supply for farms and our drinking water been elitist or not in almost everyone's best interest? Empty political rhetoric is bad in any election, but to see it in a county election is especially disappointing. Never mind substance — just make personal attacks and hope it turns voters off.
The fact that Measure 14-15 would give our community the right to democratically vote on major housing developments planned for the forestlands that provide our water makes the charge that Measure 14-15 is about the special interests of farmers or people concerned about water even more ridiculous. All Measure 14-15 does is give people the right to have a voice in deciding whether housing projects that are generally illegal in zoned forestland are worth the potential threats to farms and our drinking water.
I think it makes sense for us to have a real voice in making sure Hood River County farms have a reliable water supply and that our drinking water is safe — and that's why I'll try to ignore the politics of name calling and send in my ballot in support of Measure 14-15.
James Thompson
Mt. Hood
No on 14-15
Let's say you are arrested for a crime. You go before a judge and jury and are found "not guilty." The verdict is based on the facts and the law.
Then, the same decision is subsequently taken to a vote of the people. "Let the people decide." The people decide you are guilty and you are sent to prison for life. These people did not attend the trial, they voted by mail from home.
Anarchy is defined as the complete absence of government and the law, accompanied by political disorder.
When an applicant applies for a conditional use permit to use his or her property in a manner that complies with state and local land use law, it's wrong to say that "the people" can simply overrule the decision. A decision that has already been made by the Planning Commissioners and the Board of Commissioners based on hearings, public testimony, the facts, and the law.
That's why I'm voting no on Measure 14-15. I believe in due process and fairness.
Elizabeth Whelan
Hood River
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Parkdale third graders sing "12 Disaster Days of Christmas"
Welcome to your sing-able Christmas gift list. What follows is an emergency rendition of "12 Days of Christmas" – for outfitting your home or car in case of snow storm, earthquake, flood or other emergency. Read it as a simple list, or sing it to the tune of "12 Days" – you know, as in " … and a partridge in a pear tree…" Not to make light of it, but the song is a familiar framework for a set of gift ideas that you could consider gathering together, even if the recipient already owns items such as a bunch of coats, tire chains and flashlights. Stores throughout the Gorge are stocked up on all these items. Buying all 12 days might be prohibitive, but here are three ideas for checking any of the dozen off your list (notations follow, 1-12.) The gift items needed to stay warm, dry and safe are also coded to suggest items in your abode (A) in your car (C) or both (B). 12 Gallons of Water (A) 11 Family meals (B) 10 Cans of propane (A) 9 Hygiene bags (B) 8 Packs of batteries (A) 7 Spare coats (B) 6 Bright red flares (C) 5 Cozy blankets (B) 4 Tire chains (C) 3 Flashlights (B) 2 cell phone chargers (B) 1 And a crush-proof first aid kit (B) Price ranges? Here's a few quotes for days Three, Two, Four and Nine: n A family gift of flashlights (three will run $15-30, Hood River Supply, Tum-A-Lum) n Cell phone chargers (two will run $30-60) n Tire chains (basic set, $30, Les Schwab, returnable if unused for the winter) n Family meals ($100 or so should cover the basics for three or four reasonably well-fed days) n The home kit should be kept in a handy place near an exit, and remember that water needs to be replenished every few months. If you have a solid first aid kit already, switch out the gift idea with "and-a-sto-o-u-t- tub-for it-all …" Otherwise, it's a case of assembling your home or car kits and making sure all members of the family know what the resources are and how to use them (ie flares and propane). Emergency situations are at worst life-threatening, at best deeply uncomfortable if you and your family are left without power for an extended period, or traveling and find yourself in a situation where you need to wait out a storm, lengthy traffic delay, or other crisis. Notes on the 12 gift ideas: 12 – Gallons of water: that's one per person in a four-member family to last for three days, the recommended minimum to be prepared for utility outages. 11 – Easy-open packaged goods, energy bars, dried food and nuts are good things to include for nutrition. Think of what your family of four needs for three days to stay fortified and hydrated (see number 12). Can-opener also recommended 10 – If you have a propane camping stove, keep extra fuel handy. 9 – Hygiene bags: put packaged moistened towelettes, toilet paper, and plastic ties in large garbage bags (for personal sanitation) Resource list courtesy of Hood River County Emergency Management, Barbara Ayers, manager/ 541-386-1213. The county also reminds residents to Get a Kit, Make A Plan to connect your family if separated, and Stay Informed. See www.co.hood-river.or.us to opt-in for citizen alerts. Enlarge


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We are Americans, speaking the same language, adopting the same customs, holding the same general opinions... and shall rise and fall with Americans.
Viewing the man from the genuine abolitionist ground, Mr. Lincoln seemed cold, tardy, weak and unequal to the task. But, viewing him from the sentiments of his people, which as a statesman he was bound to respect, then his actions were swift, bold, radical and decisive. Taking the man in the whole, balancing the tremendous magnitude of the situation, and the necessary means to ends, Infinite Wisdom has rarely sent a man into the world more perfectly suited to his mission than Abraham Lincoln.
...there are at the present moment many colored men in the Confederate Army...as real soldiers, having muskets on their shoulders, and bullets in their pockets, ready to shoot down loyal troops, and do all that soldiers may do to destroy the Federal government...There were such soldiers at Manassas and they are probably there still.
There are at present many Coloured men in the Confederate Army doing duty not only as cooks, servants and labourers, but real soldiers, having muskets on their shoulders, and bullets in their pockets.
John Brown's zeal in the cause of freedom was infinitely superior to mine. Mine was as the taper light; his was as the burning sun. I could live for the slave; John Brown could die for him. The American people and the Government at Washington may refuse to recognize it for a time but the inexorable logic of events will force it upon them in the end; that the war now being waged in this land is a war for and against slavery.
Men of Color, To Arms! The case is before you. This is our golden opportunity. Let us accept it, and forever wipe out the dark reproaches unsparingly hurled against us by our enemies. Let us win for ourselves the gratitude of our country, and the best blessings of our posterity through all time.
Civil war was not a mere strife for territory and dominion, but a contest of civilization against barbarism.
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RecipesAppetizers Soups Main Dishes Sides Desserts Nutritional InformationNutritional information for fresh chestnuts |
Brioza's Chestnut Flan
In a saucepot over medium heat, melt the butter and add all of the vegetables. Sweat for 5–6 minutes. Add the chestnuts and cook an additional 2–3 minutes. Season as needed. Add the Madeira wine and just cook off the alcohol. Add the cream and simmer for 15 minutes. purée in a blender and strain through a chinois. Cool in an ice bath. Add the eggs to the chestnut cream. Butter 1–2 ounce molds and fill with the flan batter. Bake in a water bath for 25–35 minutes at 300 degrees F until set. Chef Stuart Brioza |


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Webcomic You Should be Reading: Seven Extraordinary Things
on September 8, 2011 at 8:38 PMComic's name: Seven Extraordinary Things
Creator: Doug Lefler
URL: http://www.sevenextraordinarythings.com
What is the comic about: A coming-of-age tale of a boy who makes his dreams come true, only to realize he has accomplished nothing.
How is comic drawn: black and white lineart, with heavy shadows.
How often updated: it is a completed graphic novel.
What else does the website offer: blog, art gallery, discussion forums.
What do I think of the site's overall design: Very nicely laid out. The links are easy to find, as well as individual chapters and character bios. Plus, there's a cool trailer on the site, too.
What do I like about the strip: The first thing you'll notice is the awesome black and white art style. This strip is simply gorgeous to look at, and uses light and shadow along with heavy blacks and negative space, excellently. You can tell a lot of care was put into the renderings. The story is also very human and very relatable, and you really find yourself sympathizing with the hero, even when he doesn't do the right thing. This story is very well-written
Why should YOU read it: If you're a fan of precise linework and very human stories of love found and lost, you will like this strip.


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In real estate, location is the be-all and end-all. In social media it is rankings, but can we trust them?
Posts tagged as:
Posts tagged as:
by Urs E. Gattiker on 2013/01/15 · 2 comments 5,821 views
in a analytics: rating and ranking - who's #1,a dos and don'ts
In real estate, location is the be-all and end-all. In social media it is rankings, but can we trust them?
Tagged as: benchmarking, best fashion blog, best practice, big data, buzz marketing, fashion, Milan Fashion Week, Paris Fashion week, rating and ranking, ROI, trust
CyTRAP Labs is a company offering monitoring and tracking tools for analysis of corporate blogs that are used for comparing social media stats and metrics.
> We also help companies with:1. Developing a social media strategy
2. Implementing the strategy
3. Benchmarking: Social media audit
4. Process evaluation & improvement
CyTRAP BlogRank gives you a spot on view of your corporate blog's performance (click URL)
With two decades of application inside blue chips and FT Global 500s, his pioneering work in the field of corporate blog benchmarking and the social media audit (see his books) are now a recognized gold standard.
He is known for a high energy style that combines humor, street smarts, and board room wisdom. Read more...
Social Media Audit: Help Your Bottom Line (Elsevier BWL/Mgmt) - 2014 - Autor: Urs E. Gattiker, PhD
CyTRAP Labs: Essential source for actionable metrics
© CyTRAP Labs GmbH All rights reserved.


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£550,000 Guide Price
4 Bedrooms Detached House for sale
Guide 550,000 to 600,000 constructed by monks some 300 years ago this property oozes history and charm. having seen a complete transformation the tollhouse offers a spectacular home…
£285,000 Guide Price
3 Bedrooms Cottage House for sale
Lower Dimson, Gunnislake
Video/virtual tour available for this property. a pretty cottage with lots of character set in an elevated location with far reaching views over the tamar valley from the paved terrace…
£159,950 Guide Price
2 Bedrooms Cottage House for sale
King Street, Gunnislake
A well-presented two bedroom semi-detached cottage in the heart of gunnislake boasting ample parking with a detached garage and low maintenance rear courtyard gardens. the property…
3 Bedrooms Semi Detached House for sale
Newbridge Hill, Gunnislake
Set in the middle of this cornish village on the banks of the tamar and just five miles from the towns of tavistock and callington. a charming 3 bedroom cottage which really must be…
A charming semi-detached cottage with driveway parking in the village of gunnislake. briefly comprises; lounge, modern kitchen with integrated appliances, downstairs shower room, utility…
2 Bedrooms End Of Terrace House for sale
Gunnislake, Cornwall
This end of terrace property is located in the popular village of gunnislake and has been fully refurbished. the property has been improved to a high standard, boasting a generous…
2 Bedrooms Terraced House for sale
This two bedroom mid terrace character cottage is located in the desirable area of gunnislake. accommodation comprises of kitchen/diner, lounge, two bedrooms and shower room. the property…
£130,000 Guide Price
King Street, Gunnislake, Cornwall
This cottage is sold with no onward chain and the accommodation comprises on the ground floor of an entrance porch, lounge with wood burner and stairs ascending to the first floor…
3 Bedrooms Semi Detached House
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text: Young and cute cheerleader Gigi Rivera fucks with her boyfriend. This cute young girl in a blue skirt recorded her first POV video in which guy with big cock stretches her tight inexperienced pussy. If this girl is young and not very experienced she is fucks fantastic. Her ass is oiled so that even better is sliding on the hard dick.


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Choose Between Two Options
- $200 for a two-hour sunset cruise for up to six people ($400 value)
- $225 for a Coffins Patch Reef snorkeling cruise for up to six people ($450 value)
Sunset cruises depart approximately one hour before sunset. Customers may add additional time for $100 per hour.
The snorkel cruise includes two hours of reef time and all snorkel equipment; these cruises depart at 9 a.m. and 2 p.m. daily.
Gemini Dream Charters
Gemini Dream Charters' 40-foot, six-person yacht invites passengers aboard to to soak up the beauty of the ocean waves and the Florida Keys. Groups can set sail any time for a pleasure cruise, or opt for an evening sunset cruise for a chance to watch the sun slowly submerge into its nightly bath. For those who want to check out what's going on below the surface, Gemini also partners with Keys Huka Dive to organize snorkeling and scuba cruises. Whatever journey they choose, Gemini Dream Charters supplies water and soft drinks, passengers can also either bring their own food and alcohol on board or upgrade to a variety of specialty food and drinks.


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Скачать Teaching Rebellion: Stories from the Grassroots Mobilization in Oaxaca (PM Press) бесплатно
Teaching Rebellion: Stories from the Grassroots Mobilization in Oaxaca (PM Press) Publisher: PM Press | ISBN: 1604860324 | edition 2008 | PDF | 384 pages | 10,9 mb In 2006, Oaxaca, Mexico came alive with a broad and diverse movement that captivated the nation and earned the admiration of communities organizing for social justice around the world. The show of international solidarity for the people of Oaxaca was the most extensive since the Zapatista uprising in 1994. Fueled by long ignored social contradictions, what began as a teachers' strike demanding more resources for education, quickly turned into a massive movement that demanded direct, participatory democracy. Hundreds of thousands of Oaxacans raised their voices against the abuses of the state government. They participated in marches of up to 800,000 people, occupied government buildings, took over radio stations, called for statewide labor and hunger strikes, held sit-ins, reclaimed spaces for public art and created altars for assassinated activists in public spaces. In the now legendary March of Pots and Pans, two thousand women peacefully took over and operated the state television channel for three weeks. Barricades that were built all over the city to prevent the passage of paramilitaries and defend occupied public spaces, quickly became a place where neighbors got to know each other, shared ideas and developed new strategies for organizing. Despite the fierce repression that the movement faced—with hundreds arbitrarily detained, tortured, forced into hiding, or murdered by the state and federal forces and paramilitary death squads—people are determined to make their voices heard. "Once you learn to speak, you don't want to be quiet anymore," an indigenous community radio activist said. Accompanied by photography and political art, Teaching Rebellion is a compilation of testimonies from longtime organizers, teachers, students, housewives, religious leaders, union members, schoolchildren, indigenous community activists, artists and journalists—and many others who participated in what became the Popular Assembly of the People's of Oaxaca. This is a chance to listen directly to those invested in and affected by what quickly became one of the most important social uprisings of the 21st century. |


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Moroccan Thuya Wood Peaked Stash Box with Key
FALSEOut of Stock
Preorder Available! This product is on the way and will be in stock soon. Pre-ordering will let you receive yours as fast as possible because preorders ship first!
The Moroccan Thuya Wood Peaked Stash Box with Key is currently out of stock. Don't worry though, more are already on the way and will be available soon!
Receive In-Stock Notification:-
Product Description
A very beautiful lockable stash box.The walls of this box are very solid, giving it a very high quality feel in your hand, and the deep compartment within allows plenty of space for storage.
Traditionally carved from the aromatic Thuya wood, which is unique to the western foothills of the Atlas mountain range in the Atlantic coastal region of North Africa. Local crafts-men there have been continuing their centuries old tradition of carving wooden artefacts. They use the roots of the Thuya tree to give each one of their pieces its very individual appearance. This is especially noticeable with this particular stash box, as the peaked lids offer a huge variety of swirling grains and knot patterns.
Also inside the box is a tray with three smaller fitted compartments that sits off the bottom of the main hold by over 3cm, allowing for the storage and separation of different herbs, jewellery or objects.Note:Available in 2 Sizes see drop down menu.
Technical Specifications
height 81mm length 191mm material Wood width 141mm


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Feedback AV: New Manufacturer Helps Feed America
Feedback AV launches high-margin cables, mounts and racks with portion of proceeds going to Feeding America charity.
We are sharing stories about the charitable actions of those in the custom electronics industry. Stay tuned for more charitable stories, and please share your story here.
Feedback AV is a new manufacturer in the custom electronics industry founded on two guiding principles: one business-oriented and one philanthropic.
On the business side, the Jupiter, Fla-based company aims to give its integrators/customers an active role in determining the product mix and features it carries. To achieve that mission, Feedback AV has some real star power on its side. Not only is the company owned by the folks at Audio America, a successful distributor that is part of the PowerHouse Alliance, but former Niles veterans Al Burdett and Mike "Sparky" Detmer, who in June 2012 announced he was leaving Niles, are onboard as advisors to interact with integrators and to manage the product strategy and manufacturing, which will be done with OEM relationships in China.
Feedback AV will be offering integrators high-margin products such as cables and mounts with more categories to follow. Part of its mission is honest, active and frequent communication - "feedback" - with its dealers.
"Everyone thinks they can build a better mousetrap. Well, this is their chance," says Feedback AV CEO Ryan Munder, who also is CEO of Audio America. The company will be directly soliciting dealers for feedback to design and innovate home A/V products. Munder promises the company will already have "mildly innovative product" at its launch, which is scheduled for December 1, 2012 (product will not be available to ship until January 1, 2013.)
Dealers will be incentivized with discounts and rewards for their feedback, starting with just responding to a simple "Question of the Day" on Feedback AV's website. "Our goal is to redefine the experience when purchasing electronics," says Munder.
The philanthropic part of Feedback AV involves "feeding back" America. A percentage of every sale at Feedback AV will go to the charity Feeding America, which supports more than 200 food banks across the nation. Every $1 donated to the charity equals 8 meals for low-income U.S. citizens. The charity network supplies food to 37 million Americans each year. Already, Feedback AV has donated more than 100,000 meals to the charity.
"I just want to inspire this industry and look at this business in a different light, and hopefully inspire others to give back in different capacities," says Munder.
Jason has covered low-voltage electronics as an editor since 1990. He joined EH Publishing in 2000, and before that served as publisher and editor of Security Sales, a leading magazine for the security industry. He served as chairman of the Security Industry Association's Education Committee from 2000-2004 and sat on the board of that association from 1998-2002. He is also a former board member of the Alarm Industry Research and Educational Foundation. He is currently a member of the CEDIA Education Action Team for Electronic Systems Business. Jason graduated from the University of Southern California. Have a suggestion or a topic you want to read more about? Email Jason at [email protected]
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VIZIO Releases D-Series 4K Ultra HD Smart TVs
Comcast Delays 4K Rollout Due to HDR Issues, Lagging Behind OTT and OTA
Samsung Opens Up QLED Trademark; Pushes for Open Dynamic HDR Standard
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Composure | ||
classes taijiquan baguazhang self defence qigong about us reviews a-z
What is composure?
Composure is emotional
The ability to remain emotionally stable in the event of crisis or stress can be
a valuable quality.
We encounter many things in life that might upset a person, taijiquan teaches us
to slow down and remain calm.
Inner conflict
Inner conflict prevents outer accord.
When the mind feels to be at war with the world, there can be no peace.
Peace is not a goal or target; it occurs naturally when activity stops.
Our conditioning, opinions, memories and ideas all conflict with the way things
If you cannot control
your emotions, you cannot control your money.
(Warren Buffett)
Resistance is futile
People try to shape reality to suit their own ideas, and conflict is caused by
the resistance they encounter.
Imbuing ideas with emotion creates upset.
In taijiquan we soften our minds and bodies in order to remove resistance, we
allow emotions to rise and fall of their own accord.
Most martial arts cultivate aggression. It is a natural, human, easy response to
Taijiquan does not use aggression.
It calls upon the student to be emotionally calm and composed.
This is not so easy, but is far healthier: physically, psychologically and
Composure in taijiquan
Taijiquan is often performed slowly and the emphasis is always upon relaxed,
comfortable movement.
This slowness is calming, and allows the mind to settle.
When the body naturally relaxes and sinks; the student feels grounded and
Composure in combat
Taijiquan trains stress management by encouraging a person to remain calm during
vigorous partner work.
As physical contact becomes familiar and natural, a person becomes more relaxed
when faced with conflict.
Many uncomfortable situations can be avoided by being composed.
Composure in life
Life can be very stressful and demanding.
Taijiquan and Taoism encourage people to be relaxed and patient in the face of
By avoiding unnecessary conflict, life feels easier.
A taijiquan person avoids situations by not getting into them, by dealing with
the potential before it becomes a problem.
The Art of War
pinpoints anger and greed as fundamental causes of defeat. According to Sun Tzu,
it is the unemotional, reserved, calm, detached warrior who wins, not the
hothead seeking vengeance and not the ambitious seeker of fortune.
(Thomas Cleary)
Worth reading
Emotional awareness
Neutral state
Page created
1 June 2001
Last updated
15 December 2016


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Connection Corner:
Student/DVM Networking Event
Friday, Feb. 24: 11:45 a.m. to 1:15 p.m. • Registration: Free for OVMA Student Members (maximum: 130 students); advance registration required.
You're getting a top-notch education in veterinary school, but what's life like in the real world? This event is your chance to talk to practicing veteriarians in a variety of disciplines and get answers to the questions you might not find in a classroom. This luncheon and networking power hour is a chance for students to get their questions answered, and for current veterinarians to find potential interns and contribute to the future of the veterinary community.
Advance registration for this event is required, and participants must be Members of the OVMA.
How to Participate
Student Member sign-up has closed.
Veterinarians who would like to participate may sign up on the Volunteer page.
Special Thanks
The MVC would like to recognize Silver Sponsor Hill's Pet Nutrition for supporting this event.


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The following information will assist you in planning your event at UW Bothell. This "one-stop" page offers information on campus policies, rates, event space, as well as helpful links to enable you to have a successful event. If you have questions after reading through this information, please feel free to contact the Event & Conference Services Program at 425-352-3556, email@example.com.
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