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Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular β-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular β-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures.
The Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis.
These data indicate that changes in Wnt expression per se are unlikely to be the cause of the observed dysregulation of β-catenin expression in DD.
Dupuytren's contracture or disease (DD) is a benign fibro-proliferative disease of the hand that causes permanent finger flexion contractures [1,2]. Despite its long medical history and high prevalence among Caucasians of Northern European ancestry, reportedly as high as 30–40% [3], the underlying genetic etiology of the disease remains unknown [4]. Numerous risk factors have been reported for DD, including alcoholism, trauma, diabetes, smoking, and epilepsy, but their exact role in the disease is not clear [5]. Epidemiological studies show an increased total mortality and cancer mortality rates among men with established DD [6], suggesting the pathophysiology of this disease may overlap with that of certain cancers.
β- catenin, the central component of the 'canonical' Wnt signalling pathway (herein referred to as Wnt/β-catenin) has been implicated in the pathogenesis of DD [7-9], and abnormal β-catenin levels in primary DD cell cultures have been shown to vary with specific cell culture conditions [8,9]. β-Catenin plays both a structural role, as a cadherin-binding protein in cell adhesion junctions [10,11], and a signalling role, as part of the Wnt/β-catenin pathway [12]. Wnts are a large family of lipid modified glycoproteins [13] that regulate various cellular processes important to normal embryonic development [14]. Wnts act as paracrine factors, initiating cell signalling by binding to Frizzled (Fz) receptors. The Wnt/Fz complex can then activate one of three distinct signalling pathways that control either cell fate or differentiation (Wnt/β-catenin)[14], planar cell polarity (PCP)[15], or cell adhesion (Wnt/Ca+2/PKC)[16,17]. The co-receptor LRP5/6 (lipoprotein receptor-related proteins 5 or 6) is required for Wnt/β-catenin pathway signalling [18-20]. Once activated, the Wnt/Fz/LRP complex triggers a cascade of signalling events that ultimately lead to the stabilization of a 'cadherin-free' cytoplasmic pool of β-catenin. The cytoplasmic accumulation of β-catenin results in its translocation to the nucleus where it functions as a transcriptional activator for members of the lymphoid enhancer factor/T-cell factor (Lef/Tcf) family of DNA binding proteins [21,22].
The importance of the Wnt/β-catenin signalling is underscored by its targeted disruption in human diseases. For example, several members of the Wnt/β-catenin pathway are mutated in a variety of human malignancies [23-27]. Normally, in the absence of a 'canonical' Wnt signal or an activating mutational event, the cytoplasmic 'free' pool of β-catenin becomes serine/threonine phosphorylated, ubiquitinated (Ub) and degraded in the proteasome, via an axin-based 'destruction' complex. Axin with the aid of APC (adenomatous polyposis coli) binds to β-catenin [28], which facilitates its phosphorylation [29] via a dual kinase mechanism involving CKI (casein kinase-1) and GSK-3β (glycogen synthase kinase-3β) [30-32]. CKI, which is recruited to the destruction complex by the axin binding protein diversin [33], phosphorylates β-catenin at serine 45, an important priming step required by GSK-3β to mediate β-catenin phosphorylation at threonine 41, serine 37 and serine 33. This hyper-phosphorylated form of β-catenin is then recognized by the F-box containing protein slimb/β-TrCP, a component of the E3 ubiquitin (Ub) ligase complex, and β-catenin is targeted for degradation via the 26S proteasome [34-38]. Not surprisingly, the critical serine/threonine residues of β-catenin that are phosphorylated by GSK-3β are mutational 'hot spots' in many cancers. We have previously shown that, unlike the situation in tumors, this region (exon 3) of the β-catenin gene derived from DD samples does not contain such mutations [8]. Given the proposed role of Wnt/β-catenin signalling in DD, in this paper, we set out to examine Wnt expression in DD.
Utilizing multiple approaches, we demonstrate here that multiple Wnts are expressed within patient lesions and control normal palmar fascia (PF) tissue. The pattern of Wnt expression observed in tissue samples is altered by in vitro culture method. Comparison of Wnt mRNA levels in DD and control tissues as well as examination of primary cultures of DD cells reveal that the level and type of Wnt expression is highly variable in this fibroproliferative disease with the only consistent finding being down-regulated Wnt 11 mRNA expression in disease tissue. As Wnt-11 signalling is independent of β-catenin and no other Wnt family members display consistent alteration in expression, this data suggests other, as yet unidentified, factors are dysregulating β-catenin processing in DD.
Affymetrix microarray analysis
The primary goal of this project was to determine the expression status of all Wnts expressed in DD and control palmar fascia (PF). To achieve this, surgically resected DD and control patient samples were examined using Affymetrix Microarray as described in the methods. As shown in Figure 1, 13 of the 19 Wnts (for a review of Wnt factors: http://www.stanford.edu/~rnusse/wntgenes/humanwnt.html webcite) were identified as being expressed in DD. Data indicated that the majority of Wnts detected in both DD and control were expressed at very similar levels with only Wnts 5a and 11 displaying any variance in expression between disease and control tissues.
Figure 1. Affymetrix microarray of Wnt expression. 3 DD and 3 control samples were analyzed using the Human Genome U133 Plus 2.0 Array. The data generated represents the expression analysis from all samples. The data was sorted and normalized using Genespring software. The control value (mean of three samples relative to normalization of control #1 and corresponding disease value (mean of three samples) is shown beneath each Wnt designation on the X axis. The graph displays the mean values of each group ± standard deviation.
Wnt expression profiling in vivo and in vitro using Wnt specific primers
To confirm the Microarray data and to assess the relative contribution of each of these 13 Wnts in DD and control PF, a Wnt expression profiling study was initiated utilizing Wnt degenerate primer analysis. Prior to commencing these experiments, however, it was necessary to determine the optimal samples for analysis. We have previously reported that β-catenin levels are abnormal in DD tissue but not in primary cell isolates grown in two-dimensional monolayer culture, whereas three-dimensional culture of the same primary cell lines in FPCL can recapitulate abnormal β-catenin expression [8,9]. The initial experiments involved the isolation of total RNA from surgically resected patient samples and primary cell cultures grown either in two-dimensional monolayer culture or in three-dimensional FPCL culture as described in the methods. Total RNA samples were then reverse transcribed and the cDNA templates were amplified by PCR using Wnt degenerate oligonucleotide primers (Fig. 2). Clones of the PCR amplified products were isolated, and the cloned inserts from purified plasmid DNA digested with a series of diagnostic restriction enzymes to identify the corresponding Wnt subtype as described in the methods. Clones were categorized based on the Wnt-specific DNA fragment sizes, and several representative clones from each group sequenced. The clones isolated from control and disease PF tissue displayed a similar Wnt expression pattern, with Wnt 5a, 9a, 10b, and 11 accounting for almost all of the identifiable disease and control clones detected by this approach (as shown in Table 1). Two clones representing Wnts 8b and 9b were also isolated. Comparisons of the in vivo and in vitro expression levels of the major group of Wnts (5a, 9a, 10b, and 11) were performed by RT-PCR using the Wnt-specific oligonucleotide primers as described in the methods. As shown in Figure 3, three of the four Wnts (5a, 9a and 11) were readily detectable within FPCL cultures, while only two Wnts (5a and 11) were detected in confluent monolayer cultures. Wnt5a expression levels were relatively high for both monolayer and FPCL cultures and quite similar to the levels seen in vivo. However, comparisons between the in vivo and in vitro expression levels of Wnt 9a, 10b and 11 showed significant differences. Specifically, a distinct expression hierarchy (in vivo > FPCL > confluent monolayer) was shared by Wnts 9a, 10b and 11. Wnt10b expression was very low or undetectable in the majority of the FPCL cultures and largely undetectable in the confluent monolayer cultures.
Figure 2. Degenerate primer design. Degenerative Wnt primers (designated Upper and Lower Primers) were designed using the ClustalW program with the aid of the BioEdit sequence alignment editor. Comparison of the published nucleic acid sequences for the 13 human Wnts detected by Affymetrix Microarray revealed two highly conserved regions that were used to design degenerate oligonucleotide primers for PCR amplification as shown.
Table 1. Wnt expression profiling of surgical fascia tissue specimens.
Figure 3. Comparison of Wnt expression in RNA derived from tissue, FPCL and monolayer culture. Comparisons of the in vivo and in vitro expression levels of Wnts 5a, 9a, 10b, and 11 were performed by RT-PCR using the Wnt-specific oligonucleotide primers from RNA derived from DD and PF control samples. As shown, a distinct expression hierarchy (Tissue > FPCL > monolayer) was shared by Wnts 9a, 10b and 11 with Wnts 9a and 10b being undetected in monolayer culture. Wnt5a expression levels were unaffected by culture conditions. In all cases Wnt expression was normalized to GAPDH expression and represent the mean value ± standard deviation.
Based on this data, we concluded that these in vitro culture conditions do not recapitulate in vivo Wnt expression and that only RNA isolated directly from surgical specimens would be suitable to obtain an accurate representation of Wnt expression in DD.
Wnt expression profiling in DD and control palmar fascia by reverse transcription and degenerate primer PCR analysis
In light of the results from the in vitro and in vivo studies described above, a comprehensive Wnt expression profiling study was performed on RNA isolated directly from surgical specimens utilizing reverse transcription and Wnt degenerate primer PCR analysis. PCR products (~400 bp) were subcloned into the pCR® 4-TOPO® sequencing vector and clones were isolated as described in the methods. The DNA restriction analysis was performed and a representative subgroup of clones were sequenced to confirm Wnt identity. In total, 182 clones were isolated and identified by restriction enzyme analysis to yield a representative overview of Wnt expression in DD and control palmar fascia. As shown in Table 1, Wnts 5a, 9a, 10b, and 11 accounted for all clonal isolates. Specifically, Wnts 5a, 9a, 10b, and 11 represented 18%, 19%, 14% and 49% of the control clones (n = 57), and 15%, 2%, 73% and 12% of the disease clones (n = 125), respectively (total 182).
Real Time PCR of Wnt 10b and Wnt 11 expression in DD and control palmar fascia
Wnts 10b and 11 represented the majority of clonal isolates from DD and control tissues and their representation within each group appeared to correlate with the presence of disease. To better compare the expression levels of these Wnts, we performed Real Time PCR of Wnt 10b and Wnt 11 mRNA in surgical samples of DD and normal PF control using the relative quantitation method. As shown in Figure 4, both Wnt 10b and Wnt 11 mRNA levels were significantly decreased in total RNA derived from DD cord (Mann-Whitney Test, Wnt 10b P = 0.038, Wnt 11 P = 0.014). The expression levels of Wnt10b did not reflect the representation in the clones derived from the degenerate primer analysis or the Affymetrix Microarray analysis. Wnt 11 mRNA expression, by comparison, was lower in DD tissue than control palmar fascia by all of the techniques employed. With the exception of Wnt 11, direct comparison of the Wnt expression analysis reported here indicated that the level and type of Wnt expression is not altered between DD and control PF.
Figure 4. Real time relative quantitation of Wnt 10b and Wnt 11 expression in DD vs. control. Real time relative quantitation of PCR amplicons was performed on an ABI Prism 7700 using the comparative Ct method. UPPER PANELS: Determination of relative amplification efficiencies for Wnt10b vs. β-actin and Wnt11 vs. β-actin. As shown, target and endogenous control gene products amplified with similar efficiency. The Δ CT value (CTtarget/CTendogenous control) was determined to be 0.1696 and 0.1619 for Wnt 10b/β-actin and Wnt 11/β-actin respectively. LOWER PANEL: Real time relative PCR quantitation for Wnt10b and Wnt11 mRNAs performed on a separate subset of 7 DD and 6 PF control samples derived from surgical resection. Triplicate reactions of each dilution were performed (50 μl samples) in a 96-well plate format using SDS instrumentation (Applied Biosystems) for 45 cycles. Target and endogenous control reaction were run in separate wells in triplicate at each concentration. Relative Quantitation of Wnt 10b and Wnt 11 mRNA levels in normal palmar fascia (mean Wnt 10b = 20.74 ± 8.81, Wnt 11 = 40.89 ± 12.86) were significantly (*) different to DD (Wnt 10b = 4.83 ± 2.21; P = 0.038, Wnt 11 = 7.56 ± 4.50; P = 0.014, Mann Whitney Test). Data is shown as Mean ± SEM.
Wnt expression is altered in a variety of cancers including prostate and colon carcinoma [39,40], where β-catenin accumulation and associated gene transcription are believed to contribute to the tumor growth. As we and others have previously shown that abnormal cytoplasmic accumulation of β-catenin is present in DD, we have hypothesized that β-catenin is a central molecular mediator of its pathophysiology [7-9]. As Wnt factors are the most upstream regulating factors of β-catenin levels, we have undertaken a comprehensive analysis of Wnt expression in this disease.
Of the 19 Wnts identified to-date, 13 were detected by Affymetrix Microarray analysis in both DD and PF control tissue. Normalized expression analysis indicated that the majority of Wnts were expressed at equivalent levels in both disease and control samples with only Wnt 11 and Wnt 5a displaying any suggestion of altered expression. A large standard deviation was noted in the control samples for Wnt 11 (mean 1.29, SD 0.72) and the disease samples for Wnt5a (mean 2.62, SD 1.05). The Wnt expression of the control sample derived from a patient undergoing carpal tunnel release was not significantly different from the Wnt expression of the control samples derived from palmar fascia adjacent to disease cord in all cases including Wnt 11, where it was the sample closest to the mean (data not shown). As such, none of the variability in Wnt expression was readily attributable to genetic background.
The primary advantage of the Microarray approach is the ability to sensitively screen the expression levels of a large number of mRNA transcripts in a process with low between-assay variability. This tool is designed to compare samples, in this case DD and control PF, and the readout is designed so that control readings are normalized and disease values are reported relative to that normalized value. Thus, in this study the Microarray analysis yielded an accurate and comprehensive overview of Wnt expression in the samples of DD and control PF without indicating the relative abundance of each Wnt subtype within each category. While this data indicated that both Wnt 5a and Wnt 11 expression were potentially altered in DD, it was unclear what percentage of the total Wnt signalling potential of the cells was represented by these two transcripts.
To more rigorously determine the relative contributions of the 13 Wnt transcripts detected, we therefore continued our analysis by determining the Wnt expression profile utilizing degenerate primers. Progressive multiple sequence alignment of the Wnt gene family using ClustalW [41] revealed several highly conserved regions that could be used to design degenerate oligonucleotide primers as previously described [42], that would be able to amplify all 13 Wnts identified in the microarray. This approach has the advantage of being amenable to assessing large numbers of samples with the options of restriction enzyme analysis and/or sequence level identification of the clonal isolates. Further, as each cDNA is generated from the total RNA pool, the amplified products would be predicted to be isolated at a frequency proportional to the initial abundance of the mRNA transcripts.
Initial Wnt expression profiling experiments were directed at determining the appropriate sample for analysis. This was essential, as the central hypothesis of this study was that alterations in Wnt signalling could be the primary cause of the altered cellular accumulation of β-catenin evident by immunohistochemistry of DD tissue. We have previously reported that this accumulation of β-catenin in DD was not detected by western blotting of primary cell isolates grown in two-dimensional monolayer culture [8]. Three-dimensional cultures of the same primary cell lines in Fibroblast Populated Collagen Lattices (FPCL), however, were able to qualitatively reflect in vivo β-catenin expression due, at least in part, to the addition of appropriate isometric tension [9]. As this study was inherently quantitative, it was of interest to determine the effect of isometric tension and three-dimensional FPCL culture on the level of Wnt expression and to determine how Wnt expression level in FPCL compared to Wnt expression in vivo.
The analysis of clones by restriction analysis and sequencing revealed that Wnts 5a, 9a, 10b, and 11 accounted for the vast majority of the disease and control-derived clones that could be identified by this approach. Comparisons of the in vivo and in vitro expression levels of these Wnts (5a, 9a, 10b, and 11) were performed by RT-PCR using the Wnt-specific oligonucleotide primers. Three of the four Wnts (5a, 9a and 11) were readily detectable within FPCL cultures, while only two Wnts (5a and 11) were detected in confluent monolayer cultures. Wnt 5a expression levels were relatively high for both monolayer and FPCL cultures and quite similar to the levels seen in vivo. However, comparisons between the in vivo and in vitro expression levels of Wnt 9a, 10b and 11 showed significant differences. Specifically, a distinct expression hierarchy (in vivo > FPCL > confluent monolayer) was shared by Wnts 9a, 10b and 11. Wnt10b expression was very low or undetectable in the majority of the FPCL cultures and largely undetectable in the confluent monolayer cultures.
Based on these data, we concluded that these in vitro culture conditions do not recapitulate in vivo Wnt expression and that RNA isolated directly from surgical specimens is required to obtain an accurate representation of Wnt expression in DD. A comprehensive Wnt expression profile of 182 clones was generated utilizing reverse transcription and Wnt degenerate primer PCR analysis. As shown in Table 1, Wnt 5a was evident in 15% of DD- derived clones and 18% of control derived clones, indicating that there was no difference in expression level between these groups. In contrast, the relative abundance of clones containing Wnts 10b and 11, and to a lesser extent 9b, sequences varied considerably between DD and controls. Wnt 11 was evident in 49% of control tissue – derived clones but only 12% of those derived from disease tissue, whereas Wnt 10b displayed the opposite trend being present in only 14% of control-derived clones compared to 73% of DD-derived clones. Wnt10b is recognized to primarily signal via the "canonical" pathway leading to downregulation of GSK-3β activity and cytoplasmic accumulation of β-catenin [43]. Wnt 11, while less well characterized, has been shown to be associated with the "non-canonical" cell adhesion (Wnt/Ca+2/PKC) pathway [44]. As this data could imply a shift to increased Wnt 10b expression and decreased Wnt 11 expression in DD, consistent with an increase in signalling through the canonical pathway, it was essential to independently confirm these data.
To achieve this, Wnt 10b and Wnt 11 expression were quantitated by Real Time PCR on an additional subset of 7 DD and 6 PF control samples derived directly from surgical resection. As shown in Figure 4, these results indicate that Wnt 10b expression was significantly decreased between disease and control samples. The lack of correlation between Wnt 10b expression in the Affymetrix Microarray analysis, which indicated no change between DD and control samples, the degenerate primer analysis, which indicated an increased abundance of cDNAs derived from Wnt 10b mRNA, and the decreased Wnt 10b mRNA levels revealed by Real Time PCR data, was unexpected. These contradictory findings dictate that an exclusion of changes in Wnt 10b mRNA expression in the pathophysiology of DD cannot be made at this time. Wnt 11 mRNA expression was also variable, however in this case a consistent decrease in expression between disease and control samples was evident in all of the analytical techniques employed. Wnt 11 is reported to signal through a non-canonical pathway that does not affect β-catenin accumulation [44,45]. The absence of consistent changes in the mRNA expression of any other Wnts identified in DD that could lead to a shift to canonical Wnt signalling indicates that, accounting for variability between individual samples, there is no evidence of a consistent alteration in Wnt mRNA expression in DD that would alter β-catenin accumulation.
While Wnt expression is unchanged, it should be noted that these data do not rule out alterations in Wnt signalling in the pathogenesis of this disease. Wnt signalling efficiency has been shown to be altered in a variety of tumors secondary to changes in the expression of secreted Wnt antagonists, such as the Dickkopf family, Wnt inhibitory factor-1 and secreted Frizzled-related protein (sFRP) family [46-48]. Our Affymetrix Microarray data indicated that there was a consistent reduction in sFRP-1 expression in the 3 DD samples screened relative to PF control levels (data not shown). It is possible, therefore, that alterations in total (rather than individual) Wnt signalling activity may be affected by down-regulation of sFRP-1 in DD. Importantly, post-translational modification of Wnt/β-catenin signalling pathway components, such as altered phosphorylation, acetylation, methylation, ubiquitination, sumoylation, glycosylation or lipidation, could contribute to an altered responses of DD cells to upstream signalling molecules. In addition, while we have shown that exon 3 mutations of the β-catenin gene are not evident in DD [8], we have not ruled out the possibility that changes to other regions of this or other genes encoding integral components of this pathway such as APC may be altering the sensitivity of DD cells to Wnt signalling. We are presently assessing the activity of GSK-3β in DD to determine if upstream signalling molecules are regulating kinase activity and cytoplasmic stability of β-catenin in this fibroproliferative disorder.
Wnt gene expression was shown to be affected by in vitro culture, both in routine monolayer culture and in three dimensional culture in FPCL, when assessed by RT-PCR. Affymetrix Microarray analysis, degenerate primer analysis and Real Time relative PCR quantitation were performed and, with the exception of Wnt 10b where expression was highly variable between analyses, the data indicate that overall Wnt expression is unchanged between samples derived from surgical specimens of DD and those derived from normal PF. As such, alterations in the expression levels of individual Wnt subtypes are unlikely to be contributing to the observed dysregulation of β-catenin expression in DD.
Clinical specimen collection
DD patient specimens (normal and disease PF) were collected in compliance with the University's Human Research Ethics Committee. Disease cords and nodules (disease) and uninvolved normal fascia (control) were collected from patients undergoing surgical resection of DD lesions as following: All samples used in the current study were from primary resections. In the operating room, the superficial surface of the affected palmar fascia including a surrounding area of normal appearing fascia was widely exposed. The diseased part was subsequently resected with a cuff of normal appearing palmar fascia. On the side table, the diseased cord was then removed from the resected specimen and immediately sent to the lab. In addition, an area of uninvolved normal appearing fascia (control) well-away from the resected specimen was harvested. To incorporate a "true normal", palmar fascia from patients who do not have any signs of Dupuytren's disease was harvested. Thus, one sample from a patient undergoing carpal tunnel release was also utilized in the Affymetrix Microarray study. The majority of clinical specimens were processed immediately for total RNA isolation as described below, unless otherwise indicated, in which case samples were stored at -80°C prior to processing.
Affymetrix microarray analysis
Surgical specimens were transferred to dry ice immediately after clinical dissection. Approximately 100 mg tissue samples were minced into small pieces on dry ice and then snap-frozen in Liquid Nitrogen. Tissue samples were ground in a mortar and pestle and 1 ml TRIzol reagent (Invitrogen Canada Inc., Burlington, Ontario) was added until all the powder dissolved. Samples were transferred to 1.5 ml microcentrifuge tubes and total RNA was isolated using TRIzol procedure. Total RNA was purified and stabilized using RNeasy Minikit (Qiagen Inc., Mississauga, Ontario). Aliquots (3 μl) were screened using an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, Ontario) and high quality RNA samples were submitted to the London Regional Genomic Center http://www.lrgc.ca webcite for Microarray analysis on a Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). The data generated represents the expression analysis from all samples (3 DD compared to 3 control consisting of 2 PF and 1 carpal tunnel release). The data was analyzed using Genespring software (Agilent Technologies, Mississauga, Ontario).
Primary cell monolayer culture
Primary cell cultures were established as previously described [9,49]. Initially, primary cell cultures were grown in starter media containing α-MEM (Gibco, Invitrogen Corporation), 20% fetal bovine serum (FBS, Clontech Laboratories, Palo Alto, CA), penicillin G + streptomycin sulfate, and fungizone (Gibco, Invitrogen Corporation). Established primary cell cultures were maintained in α-MEM + 10% FBS + antibiotics + fungizone at 37°C in a humidified chamber with 5% CO2.
Fibroblast populated collagen lattice (FPCL) cultures
Collagen lattices were prepared as a modified version of the method described previously [8]. Briefly, phosphate buffered solution (PBS) suspensions of primary cell cultures (passages 2 – 6) were mixed with a neutralized solution of Vitrogen100 (Collagen Corp, Santa Clara, CA, USA) collagen type I matrix (8 parts Vitrogen100, 2.9 mg/ml + 1 part 10× α-MEM + 1 part HEPES buffer, pH 9.0). The cell-collagen concentrations were adjusted with PBS to attain a final collagen concentration of 2.0 mg/ml and a final cell concentration of 105 cells/ml of matrix. The cell-collagen mixture was then dispensed into 24 well culture dishes (0.5 ml/well) that were pre-treated with a PBS solution containing 2% (w/v) bovine serum albumin (BSA). Following FPCL polymerization, 0.5 ml of growth medium (α-MEM, 10% FBS) was added on top of each lattice. After 4 days of culture the attached FPCLs were harvested for RNA extraction using the RNeasy® columns according to the manufacturer's instructions (Qiagen, Mississauga, ON, Canada).
Degenerate Wnt primer design
Progressive multiple sequence alignment of the Wnt gene family was carried out using the ClustalW program with the aid of the BioEdit sequence alignment editor [41]. Comparison of the published nucleic acid sequences for the 13 human Wnts detected by Affymetrix Microarray revealed several highly conserved regions that were used to design degenerate oligonucleotide primers for PCR amplification as previously described [42].
RNA extraction and PCR amplification of Wnt genes using degenerate oligonucleotide primers
RNA extraction from tissues was carried out as previously described. First-strand cDNA was reverse transcribed (RT) from 1 mg of total RNA using random hexamer priming and SuperScript™ II reverse transcriptase in a final volume of 50 ml, as recommended by the manufacturer (Invitrogen, Carlsbad, CA, USA). Following first strand synthesis, cDNA was amplified using the following degenerate oligonucleotide primers:
(UP) upper primer 5'-GGGGAATTCANCGAVTSYAARTGYCAY-3'
(LP) lower primer 5' – AAAAGATCTGCACCACCAVYGSWM-3'
where V = ACG, N = ACGT, R = AG, M = AC, Y = CT, H = ACT, W = AT, S = CG, K = TG and B=CGT
Standard PCR amplification was performed in a PX2 Thermal Cycler (Thermo Electron Corporation) using 4 μl of RT cDNA, 1 mM MgCl2, 200 μM of dNTPs (Invitrogen, Carlsbad, CA, USA), 1 μM of primers, and 1 unit of Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA,.USA) in Taq PCR buffer (10 mM Tris pH8.3, 50 mM KCl). PCR cycling parameters were 94°C for 5 min, followed by 40 cycles of 94°C for 1 min., 59°C for 1.5 min., 72°C for 2 min., followed by a final extension at 72°C of 10 min.
Cloning of PCR products and screening of transformants
PCR products (~400 bp) were directly subcloned into the pCR® 4-TOPO® sequencing vector using a TOPO TA cloning kit, as recommended by the manufacturer (Invitrogen). Ligation products were then used to transform TOP10 chemically competent E. coli cells, and transformants selected for using standard ampicillin (100 μg/ml) supplemented LB agar plates. Plasmid DNA was purified from selected colonies using Qiagen mini-prep columns (Qiagen, Mississauga, ON, Canada). Plasmid DNA was quantified by spectrophotometry (OD260), digested with EcoRI (pCR4-TOPO subcloned amplicons are flanked by EcoRI sites), and subjected to 2% agarose gel electrophoresis. DNA bands were gel purified (Qiagen gel extraction kit) and then digested with a series of diagnostic restriction endonuclease (RE), including Eco RI, Eco RII and Sac II. DNA fragments were then size-separated using native (non-denaturing) acrylamide (12%) gel electrophoresis. Gels were stained with ethidium bromide, placed on a UV transilluminator (FisherBiotech) and photographed using Polaroid film (type 667) and a Polaroid DS-34 camera fitted with a UV filter (Polaroid, UK). The DNA fragmentation patterns were analyzed (i.e. RE grouping), and several clones from each RE group were sequenced to confirm their Wnt identity.
RT-PCR using Wnt-specific primers
Briefly, 200 ng of total RNA from tissue or cells were reverse transcribed using random hexamers and SuperScript™ II RT as described above (Invitrogen, Carlsbad, CA, USA). Following first strand synthesis, Wnt specific PCR amplifications were carried out using 1 μl of cDNA, 1.5 mM MgCl2, 200 μM of dNTP mix (Invitrogen, Carlsbad, CA, USA), 0.8 μM of primers (see below), and 0.75 units of Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA., USA) in Taq PCR buffer (10 mM Tris pH8.3, 50 mM KCl), in a final reaction volume of 25 μl. Sequences of the Wnt-specific primer were as following:
Wnt5a upper primer (Wnt5a-UP): 5' – aagaagtgcacggagatcgt – 3'
Wnt5a lower primer (Wnt5a-LP): 5' – tggaacctacccatcccata – 3'
Wnt9a upper primer (Wnt9a-UP): 5' – gcaagcatctgaagcacaag – 3'
Wnt9a lower primer (Wnt9a-LP): 5' – tgctctcgcagttcttctca – 3'
Wnt10b upper primer (Wnt10b-UP): 5' – ctggtgctgctatgtgctgt – 3'
Wnt10b lower primer (Wnt10b-LP): 5' – cccagccaaaaggagtatga – 3'
Wnt11 upper primer (Wnt11-UP): 5' – tgacctcaagacccgatacc – 3'
Wnt11 lower primer (Wnt11-LP): 5' – tgagggtccttgagcagagt – 3'
GAPDH upper primer: 5' – gtcagtggtggacctgacct – 3'
GAPDH lower primer: 5' – aggggtctacatggcaactg – 3'
PCR cycling parameters were optimized for Wnt5a, Wnt9a, Wnt10b, Wnt11 and GAPDH to ensure log linear amplification of these products. For Wnt5a, Wnt9a, Wnt10b, Wnt11 were 94°C for 3 min, followed by 32 cycles of 94°C for 30 sec., 62°C for 45 sec., 72°C for 45 sec., followed by a final extension at 72°C of 7 min. PCR conditions for GAPDH were the same as above except 25 cycles were used. PCR samples were then subjected to 2% agarose gel electrophoresis, with the resulting amplicons being visualized by ethidium bromide staining. Digital images of the gels were captured and analyzed using a gel documentation workstation (Alpha Innotech Corp., San Leandro, CA, USA).
Real Time PCR
Real time PCR was performed on an ABI Prism 7700 (Applied Biosystems, Foster City, CA. USA) using the relative quantitation, or "comparative Ct" method. In brief, TRIzol reagent (Invitrogen) and RNeasy® Mini Kits (Qiagen, Mississauga, ON) were used to isolate total RNA from surgically resected normal and diseased tissue from patients with Dupuytren's Contracture. RNA quality was determined on an Agilent 2100 Bioanalyzer and only samples with minimal degradation were used for analysis. 10 μg of total RNA was reverse transcribed into cDNA first strand using the High-Capacity cDNA Archive Kit (Applied Biosystems) in accordance with the manufacturer's instructions. For validity, the comparative Ct method requires that the amplification efficiency of the target and endogenous control transcripts be equivalent. To assess this, dilutions of cDNA first strand corresponding to 1000 ng, 500 ng, 100 ng, 50 ng, 10 ng and 0 ng were introduced into the PCR reactions. Coupled with the target genes Wnt 10b and Wnt 11, endogenous control gene products were amplified by gene specific probe sets containing TaqMan® MGB probes labelled with 6-FAM™ (Applied Biosystems, Wnt 10b Assay Id Hs00559664_m1; Wnt 11 Assay ID Hs00182986_m1). Triplicate reactions of each dilution were performed (50 μl samples) in a 96-well plate format using SDS instrumentation (Applied Biosystems) for 45 cycles. Target and endogenous control reaction were run in separate wells in triplicate at each concentration. To determine if the target and endogenous control gene products amplified with the same efficiency, the Δ CT value (CT target/CT endogenous control) was plotted against the log input cDNA to create a semi-log regression line. A "pass" value of <0.2 for the slope of Δ CT vs. log input was used in this study. β-actin amplification passed validation with Δ CT vs. log input values of 0.1696 and 0.1619 for Wnt 10b and Wnt 11 respectively, and this control was subsequently used for all real time relative quantitation in this study.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YW, SS and RDZ carried out (RT-) PCR, DNA microarray and cell culture, as well as first interpretation of the data. DBO and BSG are responsible for study conception and design, coordinated the entire project, performed final interpretation of the data and completed the manuscript. All authors read and approved the final manuscript.
Rebecca D. Zhu is the recipient of a Canadian Institutes of Health Research Summer Research Studentship. BSG is the recipient of a Canadian Institutes of Health Research Short-Term Clinician Investigator Grant. BSG also holds a Clinician Scientist Salary Award from the Dept. of Surgery at the University of Western Ontario and a Salary Award from the Dean's Fund at the Schulich School of Medicine and Dentistry at the University of Western Ontario. Financial support for this work was provided by the Canadian Institutes of Health Research, the Lawson Health Research Institute Internal Research Fund, the US Plastic Surgery Educational Foundation, and the LHRI Advanced Surgical Technologies Research Group.
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text:
Rebuttal to the U of M's Head of Astronomy
This is a rebuttal to U. of Minnesota Astronomy Department's Mt. Graham position paper dated from Dec. 2001 The author of the U of MN position paper is Dr. Leonard Kuhi, Chair, Dept. of the Astronomy.
By the Mount Graham Coalition
P.O. Box 15451, Phoenix, AZ 85060
The University of Minnesota (UM) is considering joining the Mt. Graham telescope project.á A position paper published in December 2001 by Dr. Leonard Kuhi of the UM astronomy department contains many false or misleading statements.á This reply is to set the record straight.
ôFor reasons that come straight from the core of
the Apacheĺs rich and venerable culşture, the Apache believe that Mount
Graham is essential for maintaining their tradşitional way of life and
the intricate rhythms of their roundly sacred universe.á
The teleşscopes desecrate Mount Graham because they violate and
impugn the mountainĺs ôlifeö and all associated forms of life that have
existed for centuries on the mountain.ö
Dr. Kuhi:
Project opponents ôůhave taken the University
of Arizona (UA) to court 37 times and lost every timeůö
Jimenez from Sen. McCainĺs office called the USFS [May 17, 1989] to
say that the Senators [McCain and DeConcini] were angry over the USFS
regional decision to grant the stay in road building [to hear endangered
species appeals], and that during the briefing already scheduled there
would be some 'ass-chewing.ĺ ö
McCain and USFS Chief Robertsonůhad an understanding that the USFS would
not stand in the way and would facilitate the Mt. Graham project [despite
environmental law].ö
the outset of the meeting [May 18, 1989] McCain Ĺread the riot act.ĺ
McCain did 90% of the talking during the 1-1 1/2 hour meeting. During
McCainĺs oration he severely chastised the USFS for dragging its feet.á He said he had an understanding with the Chief [Robertson] and he
was very upset that the understanding was not being honored as evidenced
by the USFS recommendation to grant the stay.ö
this expression of anger, McCain told James Abbott [Supervisor, Coronado
N.F.] that Ĺif he did not cooperate on this project he would be the
shortest tenured Forest Supervisor in the history of the Forest Service.ĺ
concluded by stating that he perceived the stay as a delay.á He further said the staff should tell the Chief
[Robertson] that McCain believed the stay should not be approved.ĺ ô
The next morning the USFS staff got back with the Chief and ôůthe Chief
confirmed a previous understanding with McCain.öáá
Eleven years later,
the squirrel population- while overestimated due to the flawed counting
methodology- was reported to be an estimated high of 550. But it has
now dropped perilously, once again, to below 325.á
This time it is due to telescope project-induced degradation
of the squirrelĺs vulnerable forest habitat. The UA astronomers ľ who
are certainly no experts on forests or insects ľ blame only the sudden
insect infestation of the squirrelĺs food trees.á
ôSome years ago, before observatories, an Environmental
Impact Study was conducted.á The
institution wishing to build those structures in the climax forest was
warned about clear cutting even a mere 8.6 acres in the middle of this
ecosystem.á This could create
an edge effect, leading to future problems.á
Imagine living deep within a shaded, cool, moist forest, but
then suddenly being exposed constantly to direct sunlight, wind and
new drainage patterns.á Isnĺt
that exactly where a major stand of dead and dying trees exists, surrounding
the observatories?á Who is to blame, man or beetles?
[Arizona Daily Star, Tucson, Arizona, Oct.
16, 2000]
Besides McCain
intimidating the USFS into proceeding in the face of the 1989 squirrel
population crash and cone-crop failure, and the bogus exaggerated squirrel
counts, a third grave biological affront had already occurred in 1988.á That, too, was the result of the heavy-handed,
politically-muscled UA arm-twisting campaign. The Endangered Species
Act, Section 7 consultation (called a ôBiological Opinionö or BO) of
the U.S. Fish and Wildlife Service approved placement of the telescopes
on Emerald Peak even though that agency said this location would cause
far greater harm to the endangered Mt. Graham Red Squirrel.á The U.S. General Accounting Office testimony before Congress in
1990 identified that BO as ônot biologically supportableö
and that it improperly incorporated ônonbiological
information in reaching an opinion that could jeopardize a speciesĺ
existenceůö (see: ôSetting the record straight regarding false or erroneous
statements of the U. of Virginia astronomy departmentĺs ôFact Sheetö
elsewhere on this website).
Dr. Kuhi:á ôThe tribe living closest to the Mt. Graham is the San Carlos Apaches.á
In 1993 the San Carlos Tribal Council voted to remain neutral
regarding the observatory.á Individual Apache remain opposed to the project.öá
The opposition
of the San Carlos and White Mountain Apache governments and their traditional
cultural and spiritual leaders has been unrelenting, notwithstanding
UAĺs efforts to co-opt them.
The record of Apache opposition includes countless
Apache public records opposing the telescope project as compiled in
the ôRecord of Apache Oppositionö booklet.
Six San Carlos Apache Council resolutions or statements
documenting their formal opposition to the telescopes, pp. 3, 7, 16,
21, 23, 40.á Page numbers correspond
to those in the publication: Record of Apache Opposition.
Three letters about the San Carlos Apache Councilĺs
opposition to the telescopes, two written by the Council, one by UA
about the councilĺs opposition, pp. 7, 8, 9.
Three letters,
San Carlos Apache government, to U.S. Forest Service, pp. 4, 5, 24.
Four letters, San Carlos Apache Council, to German
Chancellors and/or German Parliament, pp. 12, 13, 26, 37A.
Four letters, San Carlos Apache Council, to the
Vatican, pp. 14, 15, 16, 17.
San Carlos
Apache Moccasin, July
7, ĺ92, San Carlos Tribal Council to Wash. D.C., press conference, met
Italian, German Embassies, Catholic Press, congressional offices, p.
Four letters,
San Carlos Apache Council, to Italian Parliament, pp. 20, 21, 22, 37A.
San Carlos Apache Council ôTo whom it may concernö
p. 25.
San Carlos Apache Council to Janet Reno, p. 28.
San Carlos Apache Council to Senator John McCain,
p. 29.
San Carlos Apache Tribal Council to President Clinton,
p. 30
Also in the Record
of Apache Opposition is the ôneutralityö resolution, and two letters
of lone Council members in support the project, pp. 18, 18A, 29.
the undersigned spiritual leaders of the Apache people acknowledge the
central sacred importance of Dzil Nchaa Si An (Mt. Graham) to the traditional
religious practice of the Apache.á We
oppose the Mt. Graham telescope project because it will interfere with
the ability of the traditional Apache to practice their religion.ö
In addition, the Record of Apache opposition
booklet contains copies of the several San Carlos Apache Council letters,
and the dated, signed 1995, 1996, and 1997 letters of Ramon Riley, Cultural
Resources Director of the White Mountain Apache Reservation, on pages
27, 35, 36, and 37.
Dr. Kuhi:á ôThe
White Mountain Apaches, who are further [sic] away to the north of Mt.
Graham, are more opposed to the observatory.ö
Reply:á Again, it is clear that Dr. Kuhi and the Mt.
Graham astronomers donĺt remotely understand or know anything about
the Western Apaches. It is unmistakably evident that he speaks for the
UM Astronomy department, not for the Western Apaches. The fact is, the
various Western Apache reservations are committed in their opposition
to this desecration as has been demonstrated by the copious documentation
already presented in the public record as referenced above. Dr. Kuhiĺs
manipulative claim is a non sequitur and makes about as much sense
as saying Massachusetts citizens, who are farther away from New York
City, are more opposed to the Sept. 11th terrorist attack
than closer, state of Connecticut citizens. Claiming that there would
be any real difference in the opposition to the Mt. Graham observatory
by the members of the two side-by-side reservations, shows Dr. Kuhi
has been grossly misinformed about the Western Apache people in Arizona
or their history.á
Here are some important facts of Apache history.á In 1871, the Army forced the Western Apache
peoples in the Arizona territory onto what they named the ôWhite Mountain
Reservation.öá It extended from
the Mogollon Rim on the north to and including Mt. Graham on the south.á It extended West to what is now the town of
Globe, and on the East to the New Mexico state border. Six subsequent
unilateral reductions and diversions in the Apache reservation boundary
have occurred since 1871. As a result, the Apache reservation land is
now just one half the size of the original Reservation.á
Those many reductions were executed unilaterally by decree, in
violation of the U.S. treaty with the Apache.á
Those dismemberments occurred each time settlers and their politicians
coveted more of the Apaches land for their natural resources.á This was particularly true in the case of Mt.
Graham because of its forests, pure water and adjacent fertile farmland
around its base.
In 1896, the White Mountain Reservation was arbitrarily
divided in half into northern and southern parts by a decree of the
U.S. President for administrative reasons.á
The southern half was called the ôSan Carlos Reservationö and
the northern half, the ôApache Reservation.öá
Subsequently, that northern half was renamed the ôWhite Mountain
Apache Reservation.öá The U.S.
government called the southern half of the reservation ôSan Carlosö
and the northern half ôWhite Mountain.öá
This had nothing to do with their religious, linguistic and cultural
traditions, which are universal to both Reservations. Many Apaches with
close ties to Mt. Graham ended up on the northern reservation after
that arbitrary split.
That historical truth was made clear by Ramon Riley,
the Cultural Resources Director of the White Mountain Apache, Jan. 8,
1997, in a letter to Ohio State President Gordon Gee (p. 36):
represent the White Mountain Apache Tribe in all matters pertaining
to the protection of Apache cultural and historical sites and resources.á The White Mountain and San Carlos Apache tribes
are very closely related.á Until
1897 our two tribes shared a single, undivided reservation.á We still share a language and culture and emphasize
truth, respect, honor, and humanityĺs role as caretakers for the beneficiaries
of Mother Earth.á Many of the
stories and songs that perpetuate our language and culture refer to
our four sacred mountains, Dzil Nchaa Siĺan (Mount Graham) is one of
these precious mountainsůThe simple-but-essential truth is that the
long-term health of Apache people and our cultures depend in a very
real way on the physical and visual integrity of our ancestral landscapes
and on the advent, within non-Indian society, of greater respect for
our ways.ö
Here are some of the passages which appear repeatedly
in the six San Carlos Apache Council opposition/resolutions and proclamations
from 1990 through 2001:
generations our elders have instructed us on the sacredness of Dzil
Nchaa Siĺan (Big Seated Mountain, aka Mt. Graham) and its vital importance
for maintaining the integrity of our Apache culture and traditionůö
mountain, Mt. Graham, is essential to the continued practice of physical
and spiritual healing by Apache Medicinemen/women, and to their apprenticeship
as competent traditional religious specialistsůö
proposed destruction of this mountain will contribute directly to the
destruction of fundamental aspects of traditional and spiritual life
of the Apachesůö
permanent modification of the present form of this mountain constitutes
a display of profound disrespect for a cherished feature of the Apacheĺs
original homeland as well as a serious violation of Apache traditional
religious beliefsůö
The White Mountain Apache Cultural Resources Director
stated on Nov. 8, 1995 to the Chairman of the Council of German Observatories
(p. 27):
Graham is one of the sacred mountains, one of the 4 chief mountainsůBecause
of herbsůCrown Dancers, and other power these mountains teach usůthe
observatory project has significantly harmed our already damaged culture
in a profound and almost unforgivable way.ö
The White Mountain Apache Cultural Resources Director
wrote to OSU President Gordon Gee on Jan. 8, 1997 (p. 36):
write to tell you that the project is the latest in a long, sad sequence
of threats to a unique and irreplaceable cultural system that cannot
withstand many more such assaults.á
ôI urge and implore you to maintain the distance between Ohio
State and those institutionsŚinfamous in Apache countryŚthat have, without
bothering to understand the views of traditional Apache people, sought
to discredit and minimize Apache objections to the telescopes.
accept my assurance that the proposed observatory (not to mention the
disrespectful and arrogant posture of its proponents towards the Apaches
that have voiced their opposition) is repugnant to those Apaches who
understand the importance of the mountain.á
I can also assure you that no good can come from an observatory
built on institutional arrogance and aggressive contempt for divergent
values and perspective.á In sum, please do not make more complicated
my already difficult job of protecting Apache culture: stay away from
Mount Graham and reassert Ohio Stateĺs important commitment to promoting
cultural diversity and religious freedom.ö
Dr. Kuhi:á ôMGIO (Mt. Graham International
Observatory) has a standing offer to work with those wishing access
to the area adjacent to the observatory.ö
ôThe request for access to
the site must be made in writing at least two business days prior to
the date requested for access. áThe
request should be made in the MGIO [Mt. Graham International Observatory]
office in Safford, attention John Ratje, Site Manager.
ôThe request
should include a description of the specific area for which access is
ôThe requestor and members
of their party seeking access to the site for religious purposes shall
be enrolled members of a federally recognized Tribe.
ôIn conjunction with Forest
Service policy, the requestor will have already obtained authorization
from the Forest Service to enter the Red Squirrel Refugium before submitting
their request to the MGIO offices.ö
Consider also that UA police arrested an Apache who
had been praying on the mountain in September 1997.á
UA lawyers argued in Arizona state court that he was guilty of
criminal trespass for walking down the road from the mountaintop and
should be prosecuted: see ôRecord
of Apache oppositionůö (pp. 69, 70) for the following related
newspaper stories: ôMt. Graham Ĺprayer permitĺ angers Apaches,ö Arizona Daily Star, Aug. 13, 1998; ôUA requires prayer permits for
Indians on Mt. Graham, Arizona
Republic, Aug. 15, 1998; ôHave you got your permit to pray?ö Navajo Hopi Observer, Jan. 21, 1998; ôNosie acquitted of trespassing,ö
Navajo Hopi Observer, Feb.
4, 1998).á The summit access
road, built in 1990, is entirely located on federal public land, but
UA claims it is ôtheirö road and ôtheirö domain. The UA lost this case
in court.
Dr. Kuhi:á ôThe public process to gather citizen comment was found by Federal [courts]
to be in compliance with applicable laws.ö
Since UA lobbyists and lawyers claimed that they were
exempt from NEPA, the USFS simply ignored the publicĺs commentary submitted
during the ôcitizen commentö period. Written commentary previously submitted
in 1986-87, but ignored by the USFS, clearly pointed out that the mountain
was sacred, and currently being used by the Apaches for various rituals.á UAĺs 1988 special-interest rider and the millions
UA spent in court thwarted citizen enforcement of this fundamental U.S.
cultural and environmental protection law.á
UA lawyers stated in their intervenorĺs reply in
District Court in May 8, 1992:
ôůconstruction on the first three telescopes [LBT, Vatican, Max Planck] should be commenced immediately and completed without need for, or delay that might be caused by compliance with NEPA or related statutes, including the NFMA and NHPA.ö
Judge Reinhardt to UA lawyer Todd, Dec. 13, 1990:
Dr. Kuhi:
ôThe public comment period occurred early in
the process; American Indian comment was solicited.á The Zuni and Hopi responded, and their recommendations were followed.á
The San Carlos Apaches did not respond.ö
Dr. Kuhiĺs inference is erroneous. He claims that the San Carlos
Apache did not respond to a letter sent to the San Carlos Chairman from
a research associate in the UA College of Agriculture asking for a reply
within 30 days ôearly in the process.ö It is that ôprocessö which was
woefully inadequate and deliberately managed to avoid full NHPA compliance
and to minimize Apache involvement.
The Apache elders, medicinepeople, and cultural leaders
were never notified by USFS or UA.á
Sending one letter asking for a reply within 30 days to the Apache
secular government does not meet either the black letter or the spirit
of U.S. law, including NEPA, AIRFA (American Indian Religious Freedom
Act), and NHPA.á It certainly does not meet the requirements
of the U.S. Constitution and the Apachesĺ constitutional guarantees
of religious freedom, equal protection, and due process. Nor does it
comply with the fiduciary duty that the federal government has to Native
Americans under the trust responsibility. As the U.S. Supreme Court
has explained:
a humane and self imposed policy which has found expression in many
acts of Congress and numerous decisions of this Court, [the government]
has charged itself with moral obligations of the highest responsibility
and trust.á Its conduct, as disclosed in the acts of those
who represent it in dealings with the Indians, should therefore be judged
by the most exacting fiduciary standards.ö
For further illumination, UMĺs astronomers, administrators
and trustees should read the Nov. 8, 1995 letter from the White Mountain
Apache Tribe Cultural Director, Ramon Riley, to the President of the
German Council of Astronomers.á Here
is an excerpt:
ááááááááááá ôElders will not give you answers until they
know and trust youŚuntil they see you are coming from the heart.á You have to work within the patience of elders
and not expect answers immediately.á
Everything you do must have respectůMt. Graham is one of the
sacred mountains, one of the 4 chief mountainsůBecause of herbsůCrown
Dancers, and other power, these mountains teach usůThey and the stars
guide us.
It is clear that the ôprocessö used by the UA and the
Forest Service did not comply with the recommendations of the U.S. Department
of Interiorĺs National Register
Bulletin 38: Guidelines for Evaluating and Documenting Traditional Cultural
Dr. Kuhi:
ôThe facility is not on an Indian Reservation.ö
* * *
The only way the Mt. Graham telescope project was allowed
to be placed on Mt. Grahamĺs sacred, as well as biologically irreplaceable
summit ecosystem, was by a special-interest group of Arizona astronomers
buying their way around the law. UA has spent many millions of dollars
in lobbying and pursuing in court their claim that their riders have
exempted them from all of the applicable cultural, religious and environmental
protection laws of this nation.á Is
this the sort of ethical standard a great university wishes to teach
their students, faculty, and administrators?
What one respected NOAO/AURA astronomer in Tucson said
about the Mount Graham observatory:
ôWhat has happened is all of astronomy has gotten a bad nameůThe Mt. Graham project is all about ôself-aggrandizementůItĺs got nothing to do with science, technology and truth or the best use of taxpayers money.ö [18]
[1] Apache Survival Coalition, Ola Cassadore Davis; and Ernest Victor vs. USA/USFS, U.S. District Court, Tucson, AZ April 9, 1992, Declaration of Keith Bass
[2] ôHow The UA Knocked Off Mt. Graham,ö Chuck Bowden, City Magazine, Tucson, AZ, Jan. 1989 (see elsewhere on this web page)
[3] UA lost July 29, 1994 in U.S. District Court, Tucson, and lost three other times in the U.S. Court of Appeals in San Francisco Aug. 23, 1994, Apr. 24, 1995, July 20, 1995.
UA lost in 1993 in Pima County Superior Court, for false arrest of environmental news photographer Dr. Robin Silver by UA campus police at a public demonstration opposing the Mt. Graham project.
UA lost in Maricopa County Superior Court in 1993 trying to withhold a study (the Booz-Allen Report) which revealed the UA telescope mirror lab was a financial drain and major liability to the University due toá ôunforeseen technical/production problems with 8-meter mirrors.ö They noted UAĺs loss of former clients to technically better, more competent mirror products and manufacturers. Booz-Allen also pointed out that the bonding mechanism for financing the Mt. Graham LBT Telescope may be illegal, viz. ôAttempting to use the float of non-profit bond proceeds in an interest bearing account to offset operating costs does not make good fiscal sense and may be illegal.öáá
The report showed the UA Mirror Lab had lost contracts as a result of its non-competitive product, its production/construction delays and breakdowns, and its inability to produce an 8-meter mirror on any reasonable schedule (Booz-Allen & Hamilton Inc., ôFinal Report, UA Mt. Graham Observatory Review Issues,ö Tucson AZ, Oct 23, 1991). The report also outlined a proposed UA campaign and strategy that would make ôoutliersö or outcasts of traditional Apache people who opposed the telescopes.á This, the report said, would be accomplished through a UA program of economic enticements to the San Carlos Apache tribe. See p.11 of Record of Apache Opposition to the Desecration of Mt. Graham by the University of Arizona and their Astronomer-collaborators, Nov. 2001, (available from: Mt. Graham Coalition, P.O. Box 15451, Phoenix, AZ 85060, $20.00 postpaid).
UA lost Jan. 20, 1998 in State of Arizona Court for falsely arresting a San Carlos Apache tribal member for praying on Mt. Graham.á The Tribal member had gone there to pray.
[4] National Environmental Protection Act (NEPA- a law protecting cultural and religious issues as well as environmental and endangered species issues, Endangered Species Act (ESA), National Forest Management Act (NFMA-a law protecting unique biotic communities on public lands- such as the unique, virgin, summit spruce/fir ecosystem on top of Mt. Graham and its 18 unique plants and animals found nowhere else in the world) National Historic Preservation Act (NHPA- a law protecting unique cultural, religious and/or historic sites).
[5] Internal Arizona Game and Fish Department Study: Mt. Graham Squirrel Numbers Inflated, News Advisory, Southwest Center for Biological Diversity, Dec. 2, 1997.á The AGFD report, dated July 23, 1997, confirms that agencies have been inflating red squirrel numbers in six different ways.
[6] Mt. Graham Red Squirrel extinction probability ômoderate to very high,ö News Advisory, Center for Biological Diversity, June 25, 2001 (New study ôDemographic Analysis for the Mt. Graham Red Squirrel,ö Daniel Doak, Elaine Harding, Cynthia Hartway, Dept of EEB, UC, Santa Cruz, March 5, 2001, re-confirms earlier studies warning of cumulative threats of piecemeal habitat destruction.á Study also contradicts UA claim of ôno significant effectö by telescopes).á
[7] U.S. General Accounting Office, Testimony to Congress, Statement of James Duffus III, Director, June 26, 1990, ôFWSĺ Biological Opinion Based on Other Than Biological Opinion.ö
[8] ôRecord of Apache Opposition to the Desecration of Mt. Graham by the University of Arizona and their Astronomer-collaborators,ö Nov. 2001, Mt. Graham Coalition, P.O. Box 15451, Phoenix, AZ 85060, $20.00 postpaid.
[9] Oct. 4, 1989, traditional Apache complain in both Tucson newspapers (Star, Citizen) first day of telescope deforestation (p 42); Dec. 14, ĺ89 San Carlos Apache Tribal Council (SCAT) complains to UA about telescope desecration (p. 1); Feb. 6, ĺ90 SCAT authorizes Apache Elder to ôwork in oppositionö to project; July 10, ĺ90 SCAT passes telescope opposition resolution (p.2); Aug. 31, ĺ90 SCAT Chairman to USFS opposes telescopes, threatens suit (p. 4); June 4, ĺ91 entire SCAT to USFS signs opposition to telescopes (p. 5-5A); Aug. 19, ĺ91 Apache elders file lawsuit in Federal Court (p. 6); Dec. 10, ĺ91 SCAT passes opposition resolution in front of visiting UA President (p. 7,8); Feb. 8, ĺ92 SCAT Chairman asks pope to meet Apache in Rome (p. 9); April 28, ĺ92 SCAT Chairman sends resolutions to pope opposing the Vatican telescope desecration (p. 9); April 10-28, ĺ92 Apache Medicinepeople, Spiritual Leaders sign opposition resolution (p.10); June 24-26 ĺ92, SCAT delegation to Wash. D.C. protests to German, Italian embassies, etc., hold D.C. press conference (p. 11); Aug. 24, ĺ92 entire SCAT signs protest letter to German Parliament (12, 13); Oct. 30, ĺ92 entire SCAT signs protest letter to Vatican; Sept. 30, ĺ92 SCAT invites Bishop to reservation (p. 14); May 21, ĺ93 SCAT votes repeat protest resolution (p. 16); Jan. 18, ĺ93 Apache delegations submit and have approved opposition resolutions at five National Congress of Am. Indians (p. 17); Jan. ĺ93, Feb. ĺ94, July ĺ99, Nov. 01 (p. 17, 38); SCA Tribal Cultural Advisory Comm. sign petition opposing telescopes (p. 19); Oct. 24, ĺ94 SCAT Chairman opposition letter to Italian Parliament (p. 20); Oct. 26, ĺ94 majority of SCAT reaffirm tribeĺs past opposition resolutions in protest letter to Italian govt.(p. 21); Mar. 13, ĺ95 Vice-chairman writes opposition letter to Italian Parliament; June 13, ĺ95 SCAT reaffirms opposition resolution (p. 23); July 12, ĺ95; SCAT History Program Director asks for long overdue USFS cultural studies (p. 24); Sept. 25, ĺ95 SCAT Vice-Chairman opposes UA lobbying for second exemption rider (p.26); Sept. 25, 1995 SCAT Vice-Chairman protests German telescope to Helmut Kohl (p. 26);á Oct. 29, ĺ59 traditional Apache introduce and get passed National Congress of American Indians telescope opposition (p. 17); Nov. 8, ĺ95 Cultural Director, White Mtn. Apache Reservation asks German astronomers to withdraw their desecration (p. 27); Nov. 15, ĺ95 Vice-Chairman SCAT asks Janet Reno to indict guilty USFS officials (p. 28); Nov. 15, ĺ95 SCAT Vice-Chair asks Sen. McCain to enforce long overdue USFS cultural studies (p. 29); Dec. 21, ĺ95 SCAT Vice-Chair asks Clinton to veto UA exemption rider (p. 30); May 30, ĺ95 SCAT Vice-Chair protests to Clinton on signing of UA rider (p. 33); Aug. 16, ĺ96 White Mtn. Apache Cultural Dir. write to Advisory Council on need for NHPA studies (p. 34); Jan. 8, 97 White Mtn. Apache Cultural Dir. asks OSU to stay away from Mt. Graham (p. 36); Dec. 3, ĺ97 SCAT Chairman thanks Clinton for vetoing line-item funds to Mt. Graham (p. 37); Nov. 13, ĺ97 White Mtn. Apache Cultural Dir. thanks Clinton for line-item veto of Mt. Graham funds (p. 37); July 15, ĺ99 White Mtn. Apache Tribal Council votes opposition resolution to Mt. Graham desecration; Mar. 24, ĺ99 SCAT Vice-Chair plea to President of Italy and Chancellor of Germany to halt telescope funding (p. 37A). June 5, ĺ01: SCAT votes 6th opposition declaration p. 40).
[10] July 13, ĺ93 SCAT ôneutralityö resolution (p. 18); Dec. 14, ĺ93 SCAT Chair Talgo letter to USFS supports ôneutralityö resolution (p. 18A); Dec. 5, ĺ95 Council member Belvado letter to Congress falsely stating: ôthe Tribe has never written ůletters of complaint to Government.ö In fact, there have been many letters of complaint to the Government (e.g. pp. 4, 5-5A, 24, 28, 29).
[11] ôNosie acquitted of trespassing,ö Navajo Hopi Observer, S.J. Wilson, Feb. 4, 1998)
[12] March 26, 1990, U.S. District Court, Tucson, AZ.
[13] Ibid.
Seminole Nation v. U.S., 316 U.S. 286, 296-297 (1942).
[15] Bulletin 38 provides the criteria, for identification and assessment of places of cultural significance, long recognized by ethnographers for years previous to publication, particularly in regard to Native Americans.
See, e.g., Pueblo of Sandia v. U.S., 50F.3d 856, 860 (10th Cir.1995). (USFS mailing of form letter to Council did not satisfy requirements of NHPA)
[16] U.S. Department of the Interior, U.S. Fish and Wildlife Service, Biological Opinion, Sec. 7 consultation, Mt. Graham Red Squirrel, Appendix Table 1, July 14, 1988,
NOAO/AURA (National Optical Astronomy Observatories/Association
of Universities for Research in Astronomy), after their 1984, 1987,
and other studies, concluded the clear technical superiority of Hawaii
and Chile and abandoned Mt. Graham, March 1987.á AURA included UC Oakland, Chicago, Cal Tech,
Colorado, Harvard, Hawaii, Illinois, Indiana, Iowa State, Johns Hopkins,
Maryland, MIT, Michigan, New York Stony Brook, Penn State, Princeton,
Texas, Washington, Wisconsin-Madison, and Yale. Additional institutions
specifically rejecting Mt. Graham after careful study included Harvard/Smithşsonşian
Institution in 1991, U. of Toronto in 1994, Michigan State, U. of
Pittsburgh in 1994, U. of Texas, in late 80ĺs,
U. of Chicago in late 80ĺs, and Georgia State in 1995. The City Council
of Pittsburgh passed a resolution citing: ôenvironmental problems
and the religious and cultural value of the mountain to the San Carlos
Apacheö and ôůthis project tarnishes the image of the Pittsburgh communityůö
The U. of Pittsburgh Administration, April 14, 1994, stated that ôThe
site in South Americaůwas clearly better than anywhere in the continental
U.SůThe astronomy department considered such factors as weather statistics
and seeing statisticsůö
[18] Star Gate, Phoenix New Times, by John Dougherty, June 16, 1993 (quoting Dr. Roger Lynds).

subset: commoncrawl


text:
Charles Kent Stone
Served in the Military, Army and faught for our Country in Vietnam. Died from Agent Orange at age 31. He was also a tug-boat Captain and owned his own boat. Left behind three girls: Christie, Karen ... show more
- Album: Army
- Heart Unheart
- Comment


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An operating system developed by Microsoft. Please only use this tag if the question is specific to this version of Windows.
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How do I run "Frogger 2 - Swampy's Revenge" on newer Computers
Someone gave me this game as a birthday present. Unfortunataly the installation does not even start under Windows 7, Compability Mode does not help with that. It is a Windows 95 game. How can I get it ...
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DOOM 1.9 .wad files compatibility
I have DOOM 1.9 [the shareware version] on a Windows 95 computer and I just beat Knee-Deep In The Dead. I know of a couple places to get other .wad files for DOOM, and I was wondering if all .wad ...


text:
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Our onsite restaurant, Beach House, will specialize in fresh local seafood, artisan pizza, and a large selection of craft beer on tap. So relax, unwind, and enjoy!
Holiday Inn Resort at Jekyll Island offers a unique setting for weddings and events with our oversized event lawn with panoramic views of the Atlantic Ocean!


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Home » Football NewsTwo Cardinals Pick For Athlons Big East's Breakout Players ListAugust 6, 2012 By Jon Hancock of KentuckySports.co Athlon Sports made a list of the Big East's Breakout Players for the 2012 season. They expect wide receiver DeVante Parker and defensive end Marcus Smith will be house hold names with by the end of the year, at least for Big East fans. With as much talent that Charlie Strong has put together at Louisville this year, I expect a lot of Cards will be household names with in the next year. For complete article go HERE. Excerpt: DeVante Parker, WR, Louisville – In some ways, Parker broke out last season. As a true freshman, he caught 18 passes for 291 yards and six touchdowns. His best performance came in the 24-17 loss to FIU, grabbing four receptions for 52 yards and one touchdown. With another offseason to work with quarterback Teddy Bridgewater, Parker should push for the team lead in receptions. As evidenced by his touchdown total from 2011 (6), Parker is one of the Big East's top threats in the redzone and should be Bridgewater's go-to guy around the goal-line once again. Marcus Smith, DE, Louisville – The Cardinals finished 10th nationally against the run last season, and this unit could be even better in 2012. Charlie Strong and coordinator Vance Bedford have accumulated depth and athletic ability, molding Louisville's line into one of the best in the Big East. Smith has bounced around the field in his career, starting at quarterback and moving to linebacker in 2010. He moved to defensive end in 2011 and made five starts last season, recording 12 tackles and 5.5 sacks. Smith should be Louisville's top pass rusher and will have an increased role in the defense in 2012.
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Anisotropic-They are magnets that have an easy axis of magnetization, so that the magnetic characteristics are optimal in one direction.
Air Gap-the air or nonmagnetic material between magnetic poles.
B (Magnetic Induction)-is the magnetic flux per unit area normal to the direction of the magnetic path. It is measured in Gauss, in the cgs system of units.
Bd (Remnant Induction)-magnetic induction that remains in a magnetic material after removal of an applied magnetic force.
BHmax (Maximum Energy Product)-the point on the demagnetization curve where the values of B & H are maximized. Measured in MGOe.
Bi (Intrinsic Induction)-the contribution of the magnetic material to the total magnetic induction, B. It is the vector difference between the magnetic induction in the material and the magnetic induction that would exist in a vacuum under the same field strength, H.
This relation is expressed by: Bi=B-H
Br (Residual induction)-also the flux density, it represents the maximum flux output from the given magnetic material.
Closed Circuit-exists when the external flux path of a permanent magnet is confined with high permeability materials.
Curie Temperature (Tc)-The temperature at which the magnetic alloy completely loses it's characteristics, and the material is no longer able to hold magnetization.
Demagnetization Curve-Also known as the BH Curve, it is the second/fourth quadrant of a hysteresis loop.
Eddy Currents-circulating electrical currents that are induced in electrically conductive elements when exposed to changing magnetic fields, creating an opposing force to the magnetic flux. Eddy currents can be harnessed to perform useful work (such as dampening of movement), or may be unwanted consequences of certain designs, which should be accounted for of minimized.
F (Magneto Motive Force)-the magnetic potential difference between any two points.
Ferromagnetic Material-material whose permeability is very much larger than 1 (from 60 to several thousands times 1), and which exhibits hysteresis phenomena.
Flux-condition existing in a medium subjected to a magnetizing force. This quantity is characterized by the fact that an electromotive force is induced in a conductor surrounding the flux at any time the flux changes in magnitude. The cgs unit of flux is the Maxwell.
Fringing Fields-leakage flux particularly associated with edge effects in a magnetic circuit.
Gauss-unit of magnetic induction (B), in the cgs unit of flux density. One gauss is equal to one maxwell per square centimeter, equivalent to lines per square inch, and Webers per square meter or Tesla.
Gauss Meter-instrument that measures the instantaneous value of magnetic induction, B. Its principle of operation is usually based on one of the following: the Hall effect, nuclear magnetic resonance (NMR), or the rotating coil principle.
H (Magnetic Force)-is the magnetizing/demagnetizing force per unit length at any point in a magnetic circuit. Measured in Oersteds.
Hc (Coercive Force)-is the demagnetizing force needed to reduce residual induction, B to zero, after magnetizing to saturation. Measured in oersteds.
Hci (Intrinsic Coercive Force)-measurement (in Oersteds) of its resistance to demagnetization. It is the demagnetizing force related in the material to zero after magnetizing to saturation.
Hysteresis Loop-is a closed curve obtained for a material by plotting corresponding values of magnetic induction(B), and magnetizing force(H), for ordinates & abscissa.
Irreversible Loss-a partial demagnetization of the magnet, caused by exposure to external fields(such as high/low temperatures), or other factors. These losses are recoverable by remagnetization, & they can be stabilized by partial demagnetization induced by temperature cycles or by external magnetic fields.
Isotropic-magnetic material with magnetic properties that are the same in any direction, resulting in the ability to be magnetized in any direction.
Keeper-is a piece(s)of soft iron that is placed on or between the pole faces of a permanent magnet to decrease the reluctance of the air gap and thereby reduce the flux leakage from the magnet. They are usually installed to resist demagnetization for Alnico magnets/assemblies.
Knee of the Demagnetization Curve-the point at which the BH curve ceases to be linear. All magnet materials, even if their second quadrant curves are straight line at room temperature, develop a knee at some temperature. Alnico 5 exhibits a knee at room temperature. If the operating point of a magnet falls below the knee, small changes in H produce large changes in B, and the magnet will not be able to recover its original flux output without re-magnetization.
Leakage Factor (F)-the ratio between the magnetic flux at the magnet neutral section and the average flux present in the air gap.
F=(B mA m)/(B, A g).
Leakage Flux-is the flux that is lost/leaked in the intended magnetic circuit.
Length of the Air Gap (Lg)-the length of the path of the central flux line of the air gap.
Load Line-the line drawn from the origin of the demagnetization curve with a slope of B/H, the intersection of which with the B-H curve represents the operating point of the magnet. Also see permeance coefficient.
Magnetic Circuit-an assembly consisting of some or all of the following: permanent magnets, ferromagnetic conduction elements, air gaps, and electrical currents.
Magnetic Flux(?)-the magnetic induction(B) over an area(A), when it is uniformly distributed and is normal to the area.
Magnetic Flux, or ? = BA.
Magnetic Length, The physical length of the magnet dimension which corresponds to the direction the magnet is magnetized. This may or may not be the magnet's orientation direction.
Maxwell-the unit of magnetic flux in the cgs electromagnetic system.
North Pole-the pole of a magnet which, when freely suspended, would point to the north magnetic pole of the earth. The definition of polarity can be a confusing issue, and it is often the best to clarify by using "north seeking pole" instead of "north pole" in specifications.
Oersted (Oe)-the unit of magnetic force, H, in the cgs electromagnetic system. One oersted (Oe) equals a magneto motive force (F) of one gilbert per cm of flux path.
Open Circuit Condition-when a magnetized magnet is by itself with no external flux path of high permeability material.
Orientation Direction-the direction in which an anisotropic magnet should be magnetized in order to achieve optimum magnetic properties. Also known as the "axis," "easy axis," or "angle of inclination."
Paramagnetic Material-material having a permeability slightly greater than 1.
Permeance(P)-the inverse of reluctance (R).
Permeability (µ )-term used to express vast relationships between magnetic induction (B), and the field strength (H).
Pole Pieces-ferromagnetic materials placed on magnetic poles used to shape and alter the effect of lines of flux.
Reluctance (R)-analogous to electrical resistance in a circuit. It is the quantity that determines the magnetic flux (?), resulting from a given magneto motive force (F). The relation as below:
F=magneto motive force
?=magnetic flux
Return Path-conduction elements in a magnetic circuit, which provide a low reluctance path for the magnetic flux.
Reversible Temperature Coefficient-The changes in flux caused by tempersture changes that is reversible.
Saturation-The condition where the increase in the magnetic force yields no increase in induction, & all of the elementary magnetic moments are oriented in one direction.
Search Coil-a coiled conductor, usually of known area and number of turns, that is used with a flux meter to measure the change of flux linkage with the coil.
Sintered Magnets-this magnet is produced from a compacted powder, which is then subjected to a heat operation where the full density and magnetic orientation is achieved.
Stabilization-is the exposure of a magnet to demagnetizing influences expected to be encountered in use in order to prevent irreversible losses during actual operation. Demagnetizing influences can be caused by high or low temperatures, or by external magnetic fields.
Temperature Coefficients-is a factor which describes the reversible change in a magnetic property with a change in temperature. When the temperature returns to its original point, the magnetic properties return spontaneously. It usually is expressed as the percentage change per unit of temperature.
Weber-the practical unit of magnetic flux. It is the amount of magnetic flux which, when linked at a uniform rate with a single-turn electric current during an interval of 1 second, will induce in this circuit an electromotive of force of 1 volt.


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Full-Res: W00075763.jpg | |
W00075763.jpg was taken on October 06, 2012 and received on Earth October 09, 2012. The camera was pointing toward TITAN at approximately 2,336,117 miles (3,759,616 kilometers) away, and the image was taken using the MT3 and CL2 filters. This image has not been validated or calibrated. A validated/calibrated image will be archived with the NASA Planetary Data System in 2013. For more information on raw images check out our frequently asked questions section. Image Credit: NASA/JPL-Caltech/Space Science Institute |


text:
Pronunciation: /ɪˈl(j)uːd/
Definition of illude in English:
Trick; delude: he had allowed his imagination to illude him
More example sentences
- He illuded her about his age.
Late Middle English: from Latin illudere 'to mock'.
Words that rhyme with illude
allude, brood, collude, conclude, crude, delude, dude, elude, étude, exclude, extrude, exude, feud, food, include, intrude, Jude, lewd, mood, nude, obtrude, occlude, Oudh, preclude, protrude, prude, pseud, pultrude, rood, rude, seclude, shrewd, snood, transude, unglued, unsubdued, who'd, you'dDefinition of illude in:
- US English dictionary
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Pop Culture
Downton Abbey Portrays Reality of Interfaith RelationshipsBy Gerri Miller
Go inside Season 5 Episode 9 where the story line of Atticus and Rose's interfaith relationship comes to a head.
Go To Pop CultureArticle about how a Catholic woman's decision to convert to Judaism opened up her father, born Jewish, to talking about his Jewish past for the first time.
Date: 06-11-2007Forward columnist Leonard Fein raises the possibility of secular conversion.
Date: 06-11-2007A man's personal narrative on maintaining his Christian faith in his Jewish family.
Date: 06-11-2007A personal narrative about a grandmother's conversion to Judaism after a Catholic upbringing.
Date: 06-11-2007A profile on composer Willy Schwarz that looks into his Jewish heritage.
Date: 06-11-2007Charlotte thought she was only sort of Jewish, but her friend Dale thought she belonged at the Shabbat table.
Date: 08-01-2012A personal narrative from an unmarried convert to Judaism who was godmother to her committed Catholic sister's child.
Date: 05-15-2007News article about Latino converts to Judaism.
Date: 08-13-2007A news article discussing the new guidelines for conversion passed by North America's reform rabbis.
Date: 06-06-2007Profile of Susan, a woman who was married to a Jewish man for many years but didn't decide to convert until they were divorced and she was in her '60s.
Date: 08-13-2007


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A drug dealer; primarily cocaine.
I'm going to get some coke from my skidaddy, Jim Cramer, on Wall St.
by Jared Bland
October 09, 2007
1 more definition
Top Definition
Skidaddy refers to Jim Cramer, the host of CNBC's "Mad Money With Jim Cramer"
"Are you ready Skidaddy?!?!?!" "BOOYA"
by Chris J. Bailey
October 13, 2006


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Devils Playground (2010)
Starring: Sean Pertwee Craig Fairbrass Colin Salmon Danny Dyer
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Editor's Note
When a revolutionary performance enhancer sparks a terrifying outbreak, humanity's only hope lies with the one test subject who didn't turn into a dangerous killing machine. RAK-295 is the latest drug from N-Gen industries. It was supposed to be the next big thing, but all of the 30,000 test subjects except for Angela Mills (MyAnna Burning) experience a terrifying transformation, the drug designed to make man stronger threatens to be his undoing. Incredibly, Angela seems completely immune to the drug's devastating side effects. Upon receiving word of this hopeful anomaly, N-Gen CEO Peter White (Colin Salmon) orders battle-hardened Head of Security Cole (Craig Fairbrass) to track Angela down, and get her into the lab. Meanwhile, Angela and her boyfriend Joe (Danny Dyer) are fighting for survival on the streets. When Cole shows up determined to drag Angela back to N-Gen Corporation, the future of mankind rests in the blood of one terrified woman with no place left to run.
Cast & Crew
Sean Pertwee | |
Craig Fairbrass | |
Colin Salmon | |
Danny Dyer | |
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Jaime Murray - Co-Star | |
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Shane Taylor - Co-Star | |
Bart Ruspoli - Co-Star | |
Bart Ruspoli - Screenwriter | |
Craig Conway - Co-Star | |
Jason Shepherd - Director of Photography | |
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Mark McQueen - Director |


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Event and Meeting Planning
Located on the 9th floor (Franklin), the BRC Event Team is ready to assist you in planning your event, allowing you to focus on the content of your program. For help, contact the Event Team.
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In the Sun Java System Portal Server 7.1 Administration console, click the Scheduling tab. By default, the Robot tab is enabled and shows the Scheduling Robot page. This page is where you set up the automatic search schedule for the robot. Use the attributes in the page to start and stop the robot at the times you specify.The table below lists the robot schedule attributes and their description.
Table 7–1 Robot Schedule Attributes
Attribute |
Default Value |
Description |
Start Robot Time in hours and minutes |
none selected |
This is the time that the robot starts to search. You need to specify the robot start time to schedule. The format is hh : mm : am or pm. For example, 12 : 00 : am. |
Days |
none selected |
Sun, Mon, Tue, Wed, Thu, Fri, or Sat Select at least one day. |
Stop Robot Time in hours and minutes |
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If you plan to run the robot continuously, it is recommended that you stop and restart it at least once per day. This gives the robot a chance to release resources and re-initialize itself. You need to specify the time in the format: hh : mm : am or pm. For example, 12 : 00 : am. |
Days |
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The Forerunner 405CX is the evolution of GPS-enabled training. This sleek sport watch tracks your distance, pace and heart rate, then wirelessly sends the data to your PC for later analysis. The 405CX features heart rate-based calorie computation and comes with a second wrist band option suitable for smaller wrists.
*This product is self verified
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