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Neurobiol Lipids 1, 7 (14 March 2003)
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Original Research: 32nd Society for Neuroscience Annual Meeting Proceedings article#:
Franck Dufour,CA Wei-Qin Zhao, Lakshmi Ravindranath, Daniel L. Alkon
Blanchette Rockefeller Neurosciences Institute, Rockville, MD, USA
email: email@example.com
This article was edited by Bruce Teter
Accepted for publication 5 November, 2002; Published
online: 14 March, 2003 | Article
Copyright © 2003 F Dufour et al., Licensee Neurobiology of Lipids
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[Original SFN Abstract]
Beside its crucial stuctural role, caveolin undergoes determinant caveolae functions. Caveoale are membrane compartments for integration of signal-transduction pathways, and caveolin forms a scaffold to bind and concentrate several signaling molecules [5] through its scaffolding domain [11]. The functional processing of caveolin (and caveolae) is dependent on the cellular level of cholesterol [12, 13, 14, 15, 16]. The cholesterol itself regulates the expression of caveolin [17, 18] and binds it [19].
Previously, biochemical, epidemiological and genetic characterictics have linked cholesterol levels to Alzheimers disease [20, 21, 22ED+, 23ED+, 24]. In this study, we compared the expression of caveolin in fibroblasts isolated from Alzheimers patients (AD) and a control population (AC). We have shown that caveolin expression and cholesterol concentration are increased in AD caveolae cellular fractions; these alterations seem to affect signal transduction processes. We propose a new hypothesis that includes a critical role for caveolin in regulating cholesterol in Alzheimers disease.
Cell culture
The present studies were carried out with human skin fibroblasts purchased from Coriell Cell Repositories (Camden, NJ, http://locus.umdnj.edu), seeded and grown to confluence in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum in T-25 (or T-75 for caveolae isolation) culture flasks (Falcon).
Cell fractioning
Triton-soluble cell lysates were prepared from 6 AC cell lines (AG11020, AG09697, AG09603, AG09878, AG04461, AG13300; 72.5 ± 8.8 years, mean ± SD) and 6 AD cell lines (AG05810, AG09908, AG06869, AG06844, AG06264, AG06265; 67 ± 10.1, mean ± SD). All the steps were carried out at 40C. Each flask was washed twice with PBS, then cells were scraped into 0.8 ml of lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP40, 1% Triton X-100, 1% protease inhibitor cocktail, pH 7.4). The 6 flask contents were pooled and the cells were pelleted for 5 min, 1400 g (Sorvall RT7 centrifuge). The cells were resuspended in 0.5 ml of lysis buffer, then, passed through a 261/2 needle 20 times, and sonicated 7 x 15 s on ice with 1 min rest in between times (total power, 50 J/W) with an Ultrasonic Homogenizer (4710 series, Cole-Parmer Instrument Co, IL). The sonicate was centrifuged for 10 min at 8,000 g (Eppendorf 5417R centrifuge). The supernatant was collected and designated as the Triton-soluble cell lysate.
Preparation of membrane fractions
The following buffers were used: buffer 1 (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% protease inhibitor cocktail), buffer 2 (10 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 0.5% NP40, 1% Triton X-100, 1% protease inhibitor cocktail, pH 7.4). To prepare AC and AD membrane fractions we used 6 AC cell lines: AG04461, AG11020, AG09697, AG11011, AG09603, AG09158 (68.8 ± 9.2 years old, mean ± SD); and 6 AD cell lines: AG06263, AG04400, AG06844, AG06265, AG06869, AG06205 (62.5 ± 3.6 years old, mean ± SD). A membrane fraction was prepared for each cell line. All steps were carried out at 40C. Each flask was washed twice with PBS, then scraped into 0.8 ml of buffer 1. The suspension was sonicated 3 x 10 s on ice with 1 min rest in between times (total power, 50 J/W) with an Ultrasonic Homogenizer (4710 series, Cole-Parmer Instrument Co, IL). An aliquot of the sonicate was saved and designed as the whole cell fraction. The remaining sonicate was centrifuged for 1 h at 100,000 g (Beckman TL-100 ultracentrifuge). The pellet was resuspended in 100 ml of buffer 2; then, the same centrifugation was repeated. The supernatant was collected and designated as the membrane fraction (plasma and intracellular membranes).
Membrane fractions were also prepared from another set of the same cell lines previously incubated for 1 h with polyclonal anti-PrP Ab-1 (Oncogene Research Products, Cambridge, MA), and diluted in DMEM (1/10,000).
Caveolae isolation
The following buffers were used to prepare caveolae-enriched fractions: buffer A (0.25 M sucrose, 20 mM Tricine, 1 mM EDTA, pH 7.8), buffer B (0.25 M sucrose, 120 mM Tricine, 6 mM EDTA, pH 7.8), buffer C (50% OptiPrep in buffer B).
AC and AD caveolae-enriched fractions were purified at 40C as previously described [25] with slight modifications. A plasma membrane fraction was prepared from 10 T-75 flasks of confluent AC or AD fibroblasts (AD cell lines: AG06265, AG06264, AG06844, AG09908, AG06869, 64.6 ± 9.2 years, mean ± SD; age-matched AC cell lines: AG11020, AG07310, AG09697, AG04461, AG13300, AG09878 69 ± 9.0 years, mean ± SD), and each cell line was grown in a separate flask. Each flask was washed twice with 5 ml of buffer A and then the cells were scraped into 3 ml of buffer A. The 10 AC (or AD) flask volumes were combined to pellet the cells by centrifugation for 5 min, 1400 g (Sorvall RT7 centrifuge). Cells were resuspended in 1 ml of buffer A, homogenized in a 2 ml tissue grinder (20 stokes), then passed through a 261/2 needle 20 times. The suspension was transferred to a 1.5 ml Eppendorf tube and centrifuge for 10 min, 1000 g (Eppendorf 5417R centrifuge). The postnuclear supernatant fraction (PNS) was saved on ice, the pellet was resuspended in 1 ml of buffer A, then centrifuged again for 10 min at 1000 g. Both of the PNSs were combined and layered on the top of 23 ml of 30% Percoll in buffer A, and centrifuged for 30 min at 84,000 g (Beckman L8-55 ultracentrifuge, 70 Ti rotor, Beckman 355631 polycarbonate centrifuge tube). The plasma membrane fraction (PMF), a visible band at about 5 cm from the bottom of the tube, was collected and its volume was adjusted to 4 ml, before being sonicated with an Ultrasonic Homogenizer (4710 series, Cole-Parmer Instrument Co, IL). The PMF was sonicated 7 x 15 s on ice with 1 min rest in between times (total power, 50 J/W). The sonicate was mixed and homogenized with 3.68 ml of buffer C and 0.32 ml of buffer A in a Beckman 355631 polycarbonate centrifuge tube. A 20% to 10% linear OptiPrep gradient (12 ml, prepared by diluting buffer C with buffer A) was poured on the top of the sample, then centrifuged for 90 min at 52,000 g (Beckman L8-55 ultracentrifuge, SW28 swinging out bucket rotor). The top 10 ml of the gradient was collected and mixed with 8 ml of buffer C, then transferred to a Beckman 355631 polycarbonate centrifuge tube. The sample was overlaid with 4 ml of 5% OptiPrep (prepared by diluting buffer C with buffer A), and centrifuged for 90 min at 52,000 g (Beckman L8-55 ultracentrifuge, SW28 swinging out bucket rotor). The caveolae-enriched fraction was a visible opaque band present in the 5% OptiPrep overlay about 1 cm under the surface.
Gel electrophoresis and immunoblots
The level of caveolin was quantified in each cell fraction by immunoblotting. All experiments were triplicated. Initially, the same amount of proteins from each sample was boiled for 10 min with an equal volume of 2X SDS sample buffer. The samples were loaded on a SDS polyacrylamide gel to separate the proteins, before transferring them to a nitrocellulose membrane. The samples were immunoblotted with anti-caveolin (BD Biosciences - Transduction Laboratories, Lexington, KY) at 40C overnight. After several washes, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at room temperature for 1 h. Then, the signal was detected by ECL TM Western blotting detection reagents (Amersham Pharmacia Biotech Inc., NJ), and exposed to a Kodak Biomax MR Film (Eastman Kodak Company, Rochester, NY). Immunoblots were quantified by measuring the optical density with an appropriate software (UN-SCAN-IT, Silk Scientific Corporation).
Immunoprecipitation of phospho-caveolin
The level of caveolin phosphorylation was compared in AC and AD whole cell fractions, prepared from 5 AC (AG04461, AG11020, AG09697, AG11011, AG09603, 70.8 ± 8.7 years, mean ± SD) and 6 AD (AG06263, AG04400, AG06844, AG06265, AG06869, 61.6 ± 3.1 years, mean ± SD) cell lines, according to the protocol decribed above. Whole cell fractions were incubated with end-to-end rocking (40C, overnight) with anti-phosphotyrosine antibodies (PY-20, Transduction Laboratories, Lexington, KY) in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% protease inhibitor mixture (Sigma, St. Louis, MO), and 1% phosphatase inhibitor mixture (Sigma, St. Louis, MO). Protein A and G (1/3 ratio respectively) agarose beads were added to the mixture, and rocked at 40C for 2 hours. After the wash, the precipitated proteins were separated by SDS-PAGE, and caveolin was detected in each sample with immunoblotting assays. Sample proteins were quantified to normalize the immunoblot optical density values.
AC (AG09697, AG09603, AG11020) and AD (AG06844, AG06869, AG06265) fibroblasts were grown on glass coverslips to reach confluence. Cells were washed twice in pre-chilled PBS, and fixed for 30 min at room temperature with 4% formaldehyde (wt/vol) in PBS. After fixation, cells were washed 3 times with PBS at room temperature for 10 min, then permeabilized with 0.1% Triton X-100, 10% normal horse serum, made in PBS, at room temperature for 30 min. Cells were washed in PBS, incubated for 2 h at room temperature with anti-caveolin (BD Biosciences - Transduction Laboratories, Lexington, KY) diluted 1/500 in PBS, and washed again before the incubation with fluorescein anti-rabbit IgG diluted in PBS 1/400 (1.25 mg/ml) for 1 h at room temperature in the dark. Finally, cells were washed and coverslips were mounted with a drop of Vectashield mounting medium, and observed using a Nikon Eclipse E800 microscope (FITC filter). Immunofluorescence measurements was also carried out on the same cell lines previously incubated for 1 h with polyclonal anti-PrP Ab-1, prepared in DMEM (1/10,000).
Protein and cholesterol assays
Protein concentrations were determined by the Micro BCATM Protein Assay Reagent Kit (Pierce, Rockford, IL). Cholesterol concentrations were quantified by the Amplex® Red Cholesterol Assay Kit (Molecular Probes, Eugene, OR). All assays were duplicated.
Triton-soluble cell lysate and whole cell fractions
The amount of caveolin detected in Triton-soluble cell lysate was 72% lower in AD than AC (6 pooled cell lines, experiment triplicated, p<0.01) (Figure 1a). Two bands were detected around 22 Kd, corresponding to previously described a (slowest band) and b (fastest band) isoforms of caveolin [26]. In the same preparation, the amount of cholesterol was 22% lower in AD than AC (6 pooled cell lines, experiment triplicated, p<0.05) (Figure 1b). Moreover, in a preliminary study of the cholesterol detected in AC cell lysate, we measured that 77% was esterified, and 23% was free cholesterol. In AD, we found 34% esterified and 66% free cholesterol.
The amount of caveolin was the same in AC and AD whole cell fractions,
and no difference was found in caveolin phosphorylation (data not shown).
Expression of caveolin (a) and cholesterol concentration (b) in Alzheimers and control fibroblast Triton-soluble lysate
Six Alzheimers or 6 control cell lines were pooled to prepare the lysates as described in Materials and Methods. a) 2 mg of total proteins were separated on SDS polyacrylamide gel, then transferred to a nitrocellulose membrane. The samples were immunoblotted with anti-caveolin, then with peroxidase-conjugated anti-rabbit IgG. The signal was detected by chemiluminescence and exposition to a film. Immunoblots were quantified by measuring the mean optical density on the film; the experiment was triplicated (* p<0.01). b) cholesterol concentrations were measured in 15 mg of total proteins by using a fluorometric method; the experiment was duplicated (* p<0.05).
Membrane fractions
Only the a-caveolin isoform was detected
in the plasma membrane fractions. The level of caveolin was 74% lower in
AD than in the AC plasma membrane fractions (Figure 2).
Expression of caveolin in Alzheimers and control fibroblast plasma membrane fractions
Six Alzheimers or 6 control cell lines were pooled to prepare the plasma membrane fractions as described in Materials and Methods. 0.2 mg of total proteins were separated on SDS polyacrylamide gel, then transferred to a nitrocellulose membrane. The samples were immunoblotted with anti-caveolin, then with peroxidase- conjugated anti-rabbit IgG. The signal was detected by chemiluminescence and exposition to a film. Immunoblots were quantified by measuring the mean optical density on the film; the experiment was triplicated (* p<0.001).
Caveolae fraction
In accordance with the findings of Smart and colleagues [25],
we found that caveolin was enriched in fraction 3 of the final Optiprep
gradient. This fraction was called the caveolae fraction. Only the a-caveolin
isoform was detected in this fraction. In this preparation, the caveolin
concentration was 3 times higher in AD than AC (Figure
3a). The cholesterol concentration was also higher (+41%) in the AD
caveolae fraction than in AC (Figure 3b).
Expression of caveolin (a) and cholesterol concentration (b) in Alzheimers and control fibroblast caveolae fractions
Five Alzheimers or 6 control cell lines were pooled to prepare the caveolae fractions as described in Materials and Methods. a) 0.2 mg of total proteins were separated on SDS polyacrylamide gel electrophoresis, then transferred to a nitrocellulose membrane. The samples were immunoblotted with anti-caveolin, then with peroxidase-conjugated anti-rabbit IgG. The signal was detected by chemiluminescence and exposition to a film. Immunoblots were quantified by measuring the mean optical density on the film; the experiment was triplicated (* p<0.01). b) cholesterol concentrations were measured in 4 mg of total proteins by using a fluorometric method; the experiment was duplicated (* p<0.05).
Cross-linking with anti-PrP
Incubation of AC fibroblasts with a polyclonal anti-PrP increased the
amount of caveolin found in the membrane fraction (n=6, p<0.05), (Figures
4a, 4b). The same incubation did not induce any increase of caveolin
expression in AD fibroblast membranes (Figures 4a, 4b).
Effect of anti-prion cross-linking on caveolin expression in Alzheimers and control fibroblast membrane
Six Alzheimers or 6 control cell lines were incubated with anti-prion antibodies for 1 h, then a membrane fraction was prepared from each cell line as described in Materials and Methods. 1 mg of total proteins were separated on SDS polyacrylamide gel, then transferred to a nitrocellulose membrane. The samples were immunoblotted with anti-caveolin, then with peroxidase-conjugated anti-rabbit IgG. The signal was detected by chemiluminescence and exposition to a film. Immunoblots were quantified by measuring the mean optical density on the film (* p<0.05). Figure 4a shows the expression of caveolin in Alzheimers and control fibroblast membrane in basal conditions and after incubation with anti-prion (+). Figure 4b shows the distribution of anti-prion cross-linking effect on caveolin level in Alzheimers and control fibroblast membrane fractions. Results are expressed in percentage of basal level.
A qualitative localization of caveolin in fibroblasts was performed
with immunofluorescence. In basal conditions, caveolin was mostly scattered
through the cells in AC (Figure 5A), whereas it was
more specifically located on the plasma membrane in AD fibroblasts (Figure
5C). Caveolin moved to membranes after anti-PrP incubation of AC fibroblasts
(Figure 5B). This incubation did not change the pattern
of caveolin distribution in AD fibroblasts (Figure 5D).
Immunocytochemical localization of caveolin in control (A, B) and Alzheimers (C, D) fibroblasts in basal conditions (A, C) and after anti-prion cross-linking (B, D)
Immunofluorescence localization of caveolin was performed on 3 Alzheimers and 3 control fibroblast cell lines previously incubated for 1 h with anti-prion, or in basal conditions. Panels presented in Figure 5 are representative of the observed patterns (20X magnification).
Alzheimers patient fibroblasts
The use of skin fibroblasts is based on the hypothesis that an affliction of the human brain markers and pathological processes of Alzheimers disease might also be expressed in peripheral tissues. A number of abnormalities in metabolic and biochemical processes described in AD brains have been found in cultured skin fibroblasts derived from AD patients [27]. Previous data have demonstrated a decrease in PKC activity [28, 29, 30, 31], alterations of calcium metabolism [32, 33, 34], dysfunctions of potassium channels [35] and MAP kinase signaling cascade [36] in AD fibroblasts. Thus, past findings suggest that fibroblasts might be useful to identify and test hypotheses on brain pathological mechanisms leading to Alzheimers disease. Peripheral tissues do offer the potential of avoiding variables introduced by differences in the post-mortem state of brain tissues.
Specific pattern of caveolin distribution in AD fibroblasts
We measured the basal expression of caveolin-1 in AC and AD fibroblast cellular fractions. Two bands (around 22 Kd) were detected on western blots by incubating the membranes with rabbit polyclonal anti-caveolin-1 antibodies (C 13630 from Transduction Lab.), except in the plasma membrane and caveolae fractions. These bands correspond to the 2 isoforms of caveolin-1, a slower (a) and faster (b) migrating species [3, 26]. Moreover, Fujimoto et al. (2000, Ref. 3) have reported that C 13630 antibodies react better with the a than the b isoform. This could explain the weakest signal, or the absence of the caveolin b-isoform detected here. Because both a- and b-caveolin have different roles in caveolae formation, it may be important to measure the isoform specific expression of caveolin in AC and AD cells. However, it has to be pointed out that in normal human skin fibroblasts, the a-isoform seems to be more efficient in and sufficient for forming functional caveolae [3].
Because of their lipid-enriched content, caveolae are insoluble in Triton X-100 at 40C [1, 37, 38]. We used this specific property to compare the expression of caveolin in whole cell preparations with and without caveolae (i.e., Triton-soluble cell lysate). No differences were found between AC and AD whole cell fractions (data not shown). However, in Triton-soluble cell lysate, we found a higher caveolin expression in AC than AD fibroblasts. Moreover, in the plasma membrane fraction, caveolin expression was also higher in AC fibroblasts. This finding tends to argue that in AD, caveolin is mostly segregated to caveolae. Based on these data, we inferred that caveolin might be highly concentrated in caveolae of AD fibroblasts.
To test this possibility, a detergent-free method was used for purifying caveolae [25]. In these conditions, AD caveoale fractions were effectively richer in caveolin than AC. These results suggest an AD-specific distribution among cell compartments of caveolin in fibroblasts. The detection of caveolin in AC and AD fibroblasts by immunocytochemistry is in accordance with these biochemical results. Instead of being widely dispersed through out the internal and plasma membranes as was true for AC fibroblasts, caveolin was preferentially located in aggregates that appeared as dots in AD plasma membranes. Such aggregates may correspond to clustered caveolin in caveolae. Further studies with immunoelectron microscopy will be needed to clarify this issue. Nevertheless, considering that the expression of caveolin-1 led to de novo formation of caveolae [39], a higher concentration of caveolin in a caveolae-enriched fraction could reflect a higher number of caveolae in AD fibroblasts.
The cellular localization of caveolin could depend on its phosphorylation status as previously shown [40, 41]. In the present study, however, we did not find any specific pattern of caveolin phosphorylation in AD. Thus, to understand the specific expression of caveolin in AD fibroblasts, we measured the concentration of cholesterol in Triton-soluble cell lysates and in caveolae fractions. Cholesterol plays an important role in caveolae structure and function [12, 13, 14, 16]. It binds caveolin [19] and regulates its expression [17, 18]. In caveolae fractions, the amount of cholesterol was significantly higher in AD than in AC, consistent with the observed pattern of caveolin distribution. In a preliminary experiment, the level of cholesterol in Triton-soluble cell lysate was lower in AD. Interestingly, we found that instead of 23% in AC fibroblasts, 66% of the total cholesterol was present as free cholesterol (FC) in AD. This observation could be critical for the final caveolin distribution, since the expression of caveolae, caveolin expression, and caveolin mRNA levels, are very sensitive to the FC content of the cell [17, 42], and the newly formed FC is recovered mainly in caveolae [12, 43]. Thus, in AD fibroblasts, an abnormal level of FC, whatever its origin, could be responsible for the high level of caveolin sequestration in caveolae.
Signal transduction in AD fibroblasts
The subcellular distribution of several signaling molecules is restricted and regulated by their association with scaffolding proteins [44] of which caveolin is one [1, 11] (see Ref. 5 for review). Thus, we wondered if the overexpression of caveolin in caveolae fractions could affect cellular signal transduction in AD fibroblasts. Glycosyl-phosphatidylinositol (GPI) anchored proteins are sequestrated with caveolin in caveolae by antibody cross-linking [45, 46, 47, 48]. Prion protein, which is a GPI-anchored protein, has been localized in fibroblast caveolae after cross-linking [49]. We therefore tested the effect of prion cross-linking on caveolin expression in membrane fractions from AC and AD fibroblasts. In five AC fibroblast cell lines (6 tested), the incubation with a rabbit polyclonal anti-prion induced an increase of caveolin expression in membranes; no changes were observed in AD cell lines (6 tested). In AC fibroblasts, the same cross-linking stimulation changed the distribution of caveolin. In brief, prion cross-linking induced a caveolin shift from cytosol to membrane in most of the AC fibroblasts. This membrane localization of caveolin was quite similar to the caveolin localization in unstimulated AD fibroblasts. Such a concentration of caveolin in caveolae fractions might alter signal transduction in AD cells.
Previous studies have, in fact, shown abnormalities of signal transduction in AD fibroblasts [28, 32, 35, 50, 51, 52]. For instance, AD fibroblasts exhibit an enhanced response (calcium signaling) to bradykinin [33], probably due to an enhanced IP3 production in response to this neuropeptide [53], which is correlated with an up-regulation of bradykinin receptors [53]. Bradykinin receptors are G protein-coupled receptors, sequestered in caveolae after agonist-stimulation [54, 55]. Furthermore, the cellular bradykinin-stimulated calcium waves preferentially originate from caveolin-rich locations [56]. Considering these published data and the results presented in this study, the high expression of caveolin in AD caveolae fraction could participate in the enhancement of bradykinin-stimulated calcium response by facilitating the recruitment of bradykinin receptors.
Cholesterol metabolism and Alzheimers disease
It is now well accepted that cholesterol may play a crucial role in pathological processes of Alzheimers disease [23ED+, also see Related Articles]. Epidemiological studies showed that treatment with statins, cholesterol-lowering drugs initially prescribed for hypercholesterolemia, decreased the risk of developing Alzheimers disease [57ED+, 58ED+]. In lab animals, a cholesterol-enriched diet induced amyloid burden in the brain [59ED+]. APP, the amyloid precursor protein, can be processed either by a-secretase, and produce a non-amyloid Ab sequence, or, by b- and g-secretase, generating the Ab amyloid peptide. The competition between these two alternative pathways is therefore crucial to the etiology of Alzheimers disease. It has been suggested that at the cellular level, high cholesterol concentration could switch the secretase activities from the non-amyloidogenic to the amyloidogenic pathway. Cholesterol decreases the activity of the non-amyloidogenic a-secretase [60], whereas it increases the activity of the amyloidogenic b-secretase [22ED+]. Although an alteration of APP glycosylation [61], or a direct binding on a-secretase [22ED+] by the cholesterol were proposed, no clear mechanism can yet explain the modulation of APP metabolism by cholesterol.
Here, we suggest an alternative hypothesis to understand the role of
cholesterol in Alzheimers disease (Scheme 1), in
which caveolin would play a pivotal role. Caveolin upregulation activates
cleavage of APP in AD astrocytes [10]. We suggest
that the accumulation of (free?) cholesterol would induce an overexpression
of caveolin in caveolae compartments. It was demonstrated that caveolin
binds PKC [62], which is located in caveolae [63,
and inhibits its activity [62,
PKC activity has been reported to be weaker in AD cells [31].
An important role of a-secretase pathway activation
has been attributed to PKC [67,
In our hypothesis, the overexpression of caveolin would dramatically inhibit
PKC. Such a process would result in an underactivation of a-secretase,
and a consequent, progressive switch to the amyloidogenic pathways in Alzheimers
disease. PKC has been involved in memory processes [69],
and the initial inhibition of PKC proposed in our model could contribute
to memory alteration which is shown in the early stages of Alzheimers
disease; this model would also explain the progressive formation of amyloid
plaques. Further investigations are planned to test this hypothesis.
Hypothesis including a critical role for caveolin in regulating APP metabolism in Alzheimers disease
The overexpression of caveolin would dramatically inhibit PKC, a well-known mechanism. Such a process would result in an "underactivation" of alpha-secretase pathway, and a consequent switch to the amyloidogenic pathways in a progressive process, as in Alzheimers disease. PKC has been involved in memory processes, and the initial inhibition of PKC proposed in our model could contribute to memory alteration which is shown in the early stage of Alzheimer's disease; this model would also explain the progressive formation of amyloid plaques.
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39. Fra AM, Williamson E, Simons K, Parton RG. De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin. Proc Natl Acad Sci USA.92, 8655-9 (1995) [ PubMed ][ Back2Text ].
40. Nomura R, Fujimoto T. Tyrosine-phosphorylated caveolin-1: immunolocalization and molecular characterization. Mol Biol Cell. 10, 975-86 (1999) [ PubMed ][ Back2Text ].
41. Aoki T, Nomura R, Fujimoto T. Tyrosine phosphorylation of caveolin-1 in the endothelium. Exp Cell Res. 253, 629-36 (1999) [ PubMed ][ Back2Text ].
42. Fielding CJ, Fielding PE. Cholesterol and caveolae: structural and functional relationships. Biochim Biophys Acta. 1529, 210-22 (2000) [ PubMed ][ Back2Text ].
43. Fielding PE, Fielding CJ. Plasma membrane caveolae mediate the efflux of cellular free cholesterol. Biochemistry.34, 14288-92 (1995) [ PubMed ][ Back2Text ].
44. Faux MC, Scott JD. Molecular glue: kinase anchoring and scaffold proteins. Cell. 85, 9-12 (1996) [ PubMed ][ Back2Text ].
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46. Sargiacomo M, Sudol M, Tang Z, Lisanti MP. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 122, 789-807 (1993) [ PubMed ][ Back2Text ].
47. Harder T, Scheiffele P, Verkade P, Simons K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol. 141, 929-42 (1998) [ PubMed ][ Back2Text ].
48. Verkade P, Harder T, Lafont F, Simons K. Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells. J Cell Biol. 148, 727-39 (2000) [ PubMed ][ Back2Text ].
49. Ying YS, Anderson RG, Rothberg KG. Each caveola contains multiple glycosyl-phosphatidylinositol-anchored membrane proteins. Cold Spring Harb Symp Quant Biol. 57, 593-604 (1992) [ PubMed ][ Back2Text ].
50. Gibson G, Martins R, Blass J, Gandy S. Altered oxidation and signal transduction systems in fibroblasts from Alzheimer patients. Life Sci. 59, 477-89 (1996) [ PubMed ][ Back2Text ].
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52. Etcheberrigaray R, Payne JL, Alkon DL. Soluble beta-amyloid induces Alzheimer's disease features in human fibroblasts and in neuronal tissues. Life Sci. 59, 491-8 (1996) [ PubMed ][ Back2Text ].
53. Huang HM, Lin TA, Sun GY, Gibson GE. Increased inositol 1,4,5-trisphosphate accumulation correlates with an up-regulation of bradykinin receptors in Alzheimer's disease. J Neurochem. 64, 761-6 (1995) [ PubMed ][ Back2Text ].
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55. de Weerd WF, Leeb-Lundberg LM. Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled Galpha subunits Galphaq and Galphai in caveolae in DDT1 MF-2 smooth muscle cells. J Biol Chem. 272, 17858-66 (1997) [ PubMed ][ Back2Text ].
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57. Wolozin B, Kellman W, Ruosseau P, Celesia GG, Siegel G. Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arch Neurol. 57, 1439-43 (2000) [ PubMed ][ Back2Text ]; ED+ note: Beware of five correspondence items links at PubMed record . Also see Related articles collection below.
58. Jick H, Zornberg GL, Jick SS, Seshadri S, Drachman DA. Statins and the risk of dementia. Lancet. 356, 1627-31 (2000) [ PubMed ][ FullText ]; ED+ notes: Erratum in: Lancet. 357, 562 (2001) [ FullText ]; Seven correspondence items by: Birkenhager WH, Wang Ji-G, Staessen JA ; Cheung BMY , Kumana CR ; Blauw GJ, Shepherd J, Murphy MB for the PROSPER study group ; Kast RE ; Lowe G, Rumley A, Packard C, Shepherd J ; Ebrahim S, Shlomo YB, Smith GD, Whincup P, Emberson J. Dementia and statins. Lancet. 357, 880 (2001) [ FullText ][ Back2Text ] (Please note: Free access to FullText links at Lancet website requires free registration at The Lancet); Koudinov AR, Koudinova NV. Dementia, cholesterol and the soft science of dietary fat. BMJ (25 July 2001) [ FullText ].
59. Sparks DL, Scheff SW, Hunsaker JC, III, Liu H, Landers T, Gross DR. Induction of Alzheimer-like beta-amyloid immunoreactivity in the brains of rabbits with dietary cholesterol. Exp Neurol. 126, 88-94 (1994) [ PubMed ]; ED+ citations: Refolo LM, Pappolla MA, Malester B, et al. Hypercholesterolemia accelerates the Alzheimers amyloid pathology in a transgenic mouse model. Neurobiol Dis. 7, 321-31 (2000) [ PubMed ]; Koudinov AR, Koudinova NV. Modulation of cholesterol metabolism initiates Alzheimers amyloid deposition and neuronal dysfunction. Soc Neurosci Abstr. 26, 497 (2000) [ Abstract ][ FullText ]; Kuo Y, Wu C, Lin C, Kuo C, Chen S, Chen H. Brain region-dependent increases in b-amyloid and apolipoprotein E levels in hypercholesterolemic rabbits. Soc Neurosci Abstr. Session No.883.9 (2002) [ Abstract ][ Back2Text ].
60. Bodovitz S, Klein WL. Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein. J Biol Chem. 271, 4436-40 (1996) [ PubMed ][ Back2Text ].
61. Galbete JL, Martin TR, Peressini E, Modena P, Bianchi R, Forloni G. Cholesterol decreases secretion of the secreted form of amyloid precursor protein by interfering with glycosylation in the protein secretory pathway. Biochem J. 348, 307-13 (2000) [ PubMed ][ Back2Text ].
62. Oka N, Yamamoto M, Schwencke C, et al. Caveolin interaction with protein kinase C. Isoenzyme-dependent regulation of kinase activity by the caveolin scaffolding domain peptide. J Biol Chem. 272, 33416-21 (1997) [ PubMed ][ Back2Text ].
63. Smart EJ, Ying YS, Anderson RG. Hormonal regulation of caveolae internalization. J Cell Biol.131, 929-38 (1995) [ PubMed ][ Back2Text ].
64. Liu J, Oh P, Horner T, Rogers RA, Schnitzer JE. Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains. J Biol Chem. 272, 7211-22 (1997) [ PubMed ][ Back2Text ].
65. Mineo C, Ying YS, Chapline C, Jaken S, Anderson RG. Targeting of protein kinase Calpha to caveolae. J Cell Biol. 141, 601-10 (1998) [ PubMed ][ Back2Text ].
66. Taggart MJ, Leavis P, Feron O, Morgan KG. Inhibition of PKCalpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res. 258, 72-81 (2000) [ PubMed ][ Back2Text ].
67. Ibarreta D, Duchen M, Ma D, Qiao L, Kozikowski AP, Etcheberrigaray R. Benzolactam (BL) enhances sAPP secretion in fibroblasts and in PC12 cells. Neuroreport. 10, 1035-40 (1999) [ PubMed ][ Back2Text ].
68. McLaughlin M, Breen KC. Protein kinase C activation potentiates the rapid secretion of the amyloid precursor protein from rat cortical synaptosomes. J Neurochem. 72, 273-81 (1999) [ PubMed ][ Back2Text ].
69. Nogues X. Protein kinase
C, learning and memory: a circular determinism between physiology and behaviour.
Neuropsychopharmacol Biol Psychiatry. 21, 507-29 (1997)
[ PubMed
][ Back2Text ].
Please note: all links below provide no registration free access to articles
At Neurobiology of Lipids:
Simmonds MA. The emerging neurobiology of cholesterol. Neurobiol Lipids 1, 1 (2002) Available at: http://neurobiologyoflipids.org/content/1/1/
Wood GW, et al. Cholesterol and Alzheimer's disease. Neurobiol Lipids 1, 4 (2002) Available at: http://neurobiologyoflipids.org/content/1/4/
Koudinova NV, et al. Amyloid beta, neural lipids, cholesterol and Alzheimers disease. Neurobiol Lipids. 1, 6 (2003) Available at: http://neurobiologyoflipids.org/content/1/6/
Noteworthy articles on neurobiology of lipids. Neurobiol Lipids. [Noteworthy page].
At PubMed:
PubMed search for 'caveolae and Alzheimer's disease' [Search result].
|This article should be cited
in the following way:
Dufour F et al. Abnormal cholesterol processing
in Alzheimers disease patients fibroblasts.
Neurobiol. Lipids
Vol.1, 7 (2003), Published online March 14, 2003, Available at: http://neurobiologyoflipids.org/content/1/7/
author: Franck Dufour, Ph.D., Blanchette Rockefeller
Neurosciences Institute, 9601 Medical Center Drive, Rockville, MD 20850,
USA ; tel: (301) 294-7177, fax: (301) 294-7007 ; e-mail: email@example.com
; URL: http://www.brni.org
#Footnote: Prior to the 32nd Society for Neuroscience Meeting (Orlando, Nov. 2-7, 2002) Neurobiology of Lipids published the editorial (2002, Vol.1, 5) that compiled the abstracts on the subject of the journal scope. Neurobiology of Lipids editors were invited to select the most noteworthy abstracts from the list of more then two hundered presentations. The short list yielded eighteen presentations. The authors of these presentations were invited to publish in NoL proceedings articles matching the content of the SFN 2002 presentations. This article represents the only editors' platinum choice presentation. The acceptance date represents the day of the article appearence at the 32nd SFN Annual Meeting in Orlando, Florida, Nov 2-7, 2002.
To review other neurobiology of lipids SFN Annual Meeting abstracts 2002 and the editors' choice shortlist please click here.
article has been cited by other articles:
Neurobiology of Aging
Gaudreault SB, Dea D, Poirier J.
Cholesterol, synaptic function and Alzheimer's disease.
Neurobiol Aging 25(6), 753-759 (July 2004)
[ PubMed ]


text:
Leading you to an educated decision.
HCF AC Cable
This cable option is typically chosen for its advantages over pipe and wire branch circuits. HCF AC cable has wires preinstalled, which means there is no need to pull the wire. Now there is a new form of healthcare cable that is a HCF Type MC cable product, HCF MCAP� cable, which offers improvements in installation productivity, cost and safety.

subset: commoncrawl


text:
August 11th, 2010
"Kevin, wake up! We have a radish."
As the Gramercy Park Hotel's rooftop vegetables were first coming into flower, its three principal gardeners made no bones about delivering midnight progress reports on their crop. A lot can go wrong for novices on standard terrain - let alone those attempting to cultivate lovage, patty pan squash and chiogga beets many stories above the streets of Manhattan - so the trio make a point of sharing their triumphs as well as the losses.
The pair began scouring the historic building, recently overhauled by Ian Schraeger, to reclaim whatever materials they could find - a duck roaster, old flooring, wine boxes, a paper shredder, linens - to transform the barren space into viable space to grow vegetables, herbs and fruit for the club and restaurant downstairs.
After securing some cash, and the enthusiastic partnership of Executive Chef Nick Anderer, the newly-minted farmers began delving into the world of seeds, many from online resources like Seed Savers Exchange. They faced a dual challenge: could they indulge their collective passion for unusual, rarely seen specimens like cucumelons and red okra, while also taking into account the high wind conditions, water needs and extreme heat of a New York City summer, so high above the ground?
Undaunted, Dilworth, Denton and Anderer harvested a battered shredder, disused wine buckets and old sauce pots as planters for the seedlings they'd cultivated in cut-apart water bottles. They then fashioned an irrigation system that feeds rainwater from the building's downspout into a reclaimed 55-gallon grease drum, and into beds lined with old tablecloths from the restaurant.
They knew they were onto something when the bugs arrived of their own accord - first aphids, then a curative crew of ladybugs to tend to the invasion. The others, they imported. Namely 1,500 worms that break down the building's vegetable waste in compost bins made of repurposed flooring and the top of a duck roaster left over from the restaurant's previous incarnation as Wakiya, an upscale Chinese venue.
It's an incredible amount of constant tending, but the opportunity to serve lovingly hand-grown produce is paramount to the three restaurant veterans. "We don't grow 100% of what we serve," says Denton, "but we serve 100% of what we grow."
Dilworth is delighted with the garden's progress and is constantly dreaming of expansion plans, as he know this opportunity is rare in the New York City restaurant world. "Serving someone a pea you just picked, with the shoot on one end and the flower on the other? You just can't buy that."
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The Prefect of the Congregation for the Doctrine of the Faith has protested against the media's portrayal of him, and talks about the good relations he has with Pope Francis, reports Vatican Insider.
Just a few days before the Consistory that will create him cardinal and after months of controversies in the media over his seemingly rigid stance on pastoral care for remarried-divorced persons, Archbishop Gerhard Müller has been eager to stress that he is on good terms with Pope Francis.
'I am not his conservative opponent,' he said in an interview published by German Catholic news agency Kathpress.
The German prelate whom the Pope appointed as leader of the dicastery in charge of Church doctrine, is not happy about being presented as an internal opponent of Francis' pontificate. Müller accepts there are differences in terms of formation and approach but 'these complement each other, they are not contradictory.' The new cardinal stated that Francis' magisterium and speeches cover the 'entire Catholic faith.'
The Prefect of the Congregation for the Doctrine of the Faith meets the Pope two or three times a month and the two communicate in Italian or Spanish (Müller speaks Spanish well, having spent quite a bit of time in Latin America).
As far as women's role in the Church is concerned, Müller said they could be given some high-ranking positions in the Vatican: not in the Congregations but in the Pontifical Councils, for example the Pontifical Council on the Family (which is led by Bishop Vincenzo Paglia) or for Health Care Workers.
FULL STORY Müller: "I'm not Francis'conservative opponent" (Vatican Insider)


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Knightley's kissing champKeira Knightley has revealed Johnny Depp kisses with more passion than Orlando Bloom.
The stunning actress gets to smooch both heartthrob actors in new film 'Pirates of the Caribbean: Dead Man's Chest' - the sequel to 'The Curse of the Black Pearl'.
Ironically, earlier this week Orlando praised Keira for her fantastic kissing technique, saying: "She's a great kisser and a lovely person. She's absolutely gorgeous."
However, the 21-year-old actress doesn't seem as impressed with her co-star's smooching skills as he is with hers.
When quizzed which co-star was the best kisser, she told InStyle magazine: "Johnny Depp certainly wasn't bad."
Despite Orlando's gushing praise for Keira's kissing technique, he did admit he found it "peculiar" locking lips with the 'Pride and Prejudice' star, because they are such good friends.
He said: "Kissing scenes are a bit peculiar. It's like you play at it."
Have an question? Get it answered by AskMen's guyQ.


text:
Los Angeles Kid Lit Drink Night
Saturday, June 18 from 6-8pm
Pink Taco in the Century City Mall...meet on the patio!
BIG NEWS: LA KidLit Drink Night #2
During SCBWI SUMMER CONF
Sunday, August 7th 6pm at Pink Taco
Lee Wind just put up a Facebook Page for it:) Inspired by the Kid Lit Drink Night cooked up by Betsy Bird and Cheryl Klein in NYC, Los Angeles whips up our own version of the drop-by-for-drinks-and-food-(you-buy-your-own)-and-great-conversation-with-cool-people-in-the-Children's-Literature-Industry Evening! Masterminded by Lee Wind, Greg Pincus, Rita Crayon Huang, Sara Wilson Etienne and Jill Corcoran! Help spread the word and invite YOUR Kid Lit World friends...


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Thursday, October 25, 2012
Scott could miss Sunday's game
Veteran LB Bart Scott sat out practice for a second straight day Thursday because of a hyperextended big toe on his right foot, and revealed to reporters that he has been playing with the injury since the Jets' first game against Miami on Sept. 23. Scott is iffy for the rematch Sunday at MetLife Stadium.
"It's tough. It's like losing a thumb on your hand. Everything you do comes off your big toe," said Scott, who said he suffered the injury on the Dolphins' second play from scrimmage in Week Three.
"I've always prided myself on being able to have a high pain tolerance and to be able to play through things," said Scott, who has played in 119 straight regular-season games since 2004. "I just want to show my teammates that I'm willing to bite down and continue to play and do what I can to help us win."
"If we have to shut it down, we shut it down," he added. "If not, then we'll be ready to go."
Although Scott admitted "I've always prided myself on my toughness" and that he considers it important to go to work everyday, whether it be practices or games, he added he'll do "whatever is best for the team. I don't care about a streak. I care about helping my team win and trying to win a championship. That's always been my primary goal, and that's why I came here. If helping the team best is for me to be on the sideline and help these young linebackers see what's going on and help slow the game down for them, then I'll do that. If it's biting down so we can get to the bye at 3-1 in our division, then that's what I'll do."
If he does sit out, the thinking is that the bye week following the Miami game will make it possible for him to heal somewhat before the game at Seattle on Nov. 11.


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Illinois Gov. Quinn facing ongoing investigation
CHICAGO – Democratic Gov. Pat Quinn, locked into a competitive re-election bid, is fighting to maintain an image as a reformer who's cleaned up state government as questions about a now-defunct anti-violence program he started in the run-up to his 2010 election threaten to hang over his campaign for months.
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Illustration: Matthew Golding.
THE massacre of innocents at a primary school in Newtown, Connecticut, on Friday was indeed, as President Barack Obama put it, heart-breaking. His halting, grieving address to the nation captured the pain that is felt in the immediate aftermath of such a shocking event. But the pain will not last, for it has happened before - and no American politician will have the nerve to propose the only cure to this repetitive insanity, which would be a sensible, mature and responsible attitude towards the ownership and use of guns. They would never be elected to public office again.
This President cannot stand for election a third time, so perhaps he might be able to break the mould. But it is hard to believe. The truth is, America is a society obsessed with guns and addicted to their repulsive but effective use as instruments of death. They like them; they teach their children to shoot them, as Nancy Lanza did with her son, Adam, the young man responsible for the carnage in Connecticut; they store them; they harbour a fetish for them, and will never tolerate any attempt to deny them their toys. They rarely admit what their guns are designed to do, which is to kill.
Statistics may be a bad excuse for prose, but those that pertain to this subject make chilling and necessary reading. They demonstrate that there is a pathological pleasure at large in the United States. There are nearly 300 million guns to be found there, one-third of them handguns - which are useless for hunting purposes, but brilliant as tools for killing. This represents the highest concentration of private ownership of murder weapons in the entire world.
Every year, 17,000 people are killed in America, 70 per cent of them with guns, and nearly 20,000 people commit suicide by shooting themselves to death in the home - where a gun is readily to hand. Almost half of all US households have a gun stored as easily as the knives and forks. In the whole world, only Colombia has a worse record.
The statistics are even more heart-breaking when applied to the young. The slaughter of children by gunfire in the United States is 25 times the rate of the 20 next largest industrial countries in the world combined. If you add them all up, since the assassinations of Robert Kennedy and Martin Luther King in 1968, well over a million Americans, children and adults, have been shot to death, and even now 80 people die in this manner every day. The terrible slaughter on Friday is not as unusual as it should be.
Speaking personally, the very notion that an object in my hands could kill somebody in seconds if I pushed the right button fills me with passionate dread. Most Americans would think me blind to one obvious advantage that a gun bestows, and that is the powerful value of deterrence. Possession of a handgun protects us, they say, from random assault.
The trouble with this bland statement is that it is not true. Careful studies have demonstrated, over and over again, that homes which do not have a gun in the drawer are safer than those which do, for the mere sight of such a weapon provokes immediate action; the only thing it deters is thought.
Most tragic of all is the incidence of suicides, far higher in American homes that possess guns than in those that do not. If a gun is at hand, resolution of one's troubles can be swift and painless, effected in seconds. The gun renders suicide an attractive and emotional option; homes that harbour them are inherently dangerous places.
Adam Lanza presents us with a woeful illustration of all this. He was troubled, and he had weapons. It was easy. And yet, incredibly, suggestions have been put forward to extend the carrying of guns well beyond the home, to virtually anywhere. A Texas Republican in the House of Representatives, Louie Gohmert, once proposed a bill to permit members of Congress to carry firearms into the Capitol.
That such a barmy idea may even be uttered speaks loudly of America's malaise. In Arizona, where the attempted assassination of Congresswoman Gabrielle Giffords and the slaughter of six others took place last year, almost anyone can have a permit to carry a concealed weapon and be allowed to take guns into a bar; that same state's legislators have talked about passing laws permitting teachers and students to carry guns to school with them. Such is the contagion of madness.
Furthermore, the news channels are so far removed from any moral anguish that they happily pounce upon the scene of latest slaughter to get close-ups of bloodstains and to portray the distress of the bereaved in harrowingly stupid interviews. Some channels will not deign to air any view which might suggest that citizens, even those who are mentally disturbed, ought perhaps not to be armed with lethal weapons; they might thenceforth be refused advertising revenue if they dared.
That is why President Obama's decency, humanity and restraint should be a lesson to the media and a snub to their extravagance. In 2008, the deranged student Seung-Hui Cho mowed down 32 of his fellows at Virginia Tech. The news channels did exactly what he wanted, obliging him with massive attention. This young man had no point he wanted to prove, no statement he needed to make, no enemies that he wished to conquer. He wanted only to express himself, and took with him two powerful guns with which to do so.
This was murder in the pursuit of self-esteem, what the writer James Carroll called ''expressive violence''. It was the perversion of yet another American value, the one which says no child should have his aspirations for self-expression thwarted; ally this with the value that nobody should be denied the right to own and use a gun, and you have the loudest expression of self imaginable.
Over the decades, there have been some weak attempts at sanity, always subsequently rescinded. When a gunman tried to assassinate President Reagan and instead gravely wounded White House press secretary Jim Brady, the US Congress was shocked into action. It passed the so-called Brady Law, which included some small measures to make handguns less easily available to maniacs. In the ensuing 30 years that law has been so progressively emasculated that it now means nothing. Any politician who tried to give it some weight would not be re-elected. This is not because the public would not welcome reform, but because those with power and money would not allow it.
Here is the sort of thing they do: in Virginia, laws have been enacted prohibiting the police from mounting so-called ''sting'' operations on shops that openly sell assault weapons to people without checking their histories. The police are thereby legally prevented from doing their job of keeping public order. Whenever they try, somebody will happily go before the camera to proclaim the right to carry and use a gun without any restriction, even that of lunacy.
And here is the sort of thing they say: an Arkansas Republican called Jay Dickey told an International Herald Tribune journalist that ''it's really simple with me. We have the right to bear arms because of the threat of government taking over the freedoms that we have''. By this, the man can only mean the freedom to kill, which he cherishes higher than the freedom to be governed democratically. It is indeed a frightening remark because the US government is seen as the enemy.
Nor is this paranoia isolated. After the vile shooting in Arizona, the sale of handguns rocketed because the loonies were terrified that their government might take their guns away. Former vice-president Dick Cheney once loudly proclaimed, to wild applause and the kind of whooping with which adolescents greet pop stars or baseball heroes, that America's source of happiness sprang from the barrel of a gun. His audience, of course, was the National Rifle Association, one of the most noisome and dangerous groups in the world. It is impossible to overstate its power and influence. On the matter of weapons designed to kill, it controls America.
Members of Congress are afraid of this pernicious organisation, which has spent more than $35 million on its lobbying machine since 1997, and spends millions more on advertisements and publicity often devoted to the calumny and denigration of anyone who resists the appeal of its reckless morals. It is not above simply telling lies about its opponents to get its way.
The NRA might assume the manners and attitudes of a John Wayne, but the world which inhabits its imagination disappeared well over a century ago. Justice Stephen Breyer, with rare eloquence and clarity, has pointed out the differences between an ''18th-century, primarily rural America, where frontier life demanded guns, and the present, primarily urban America, where gun possession presents a greater risk of taking innocent lives''. This obvious truth, grounded in nothing more complex than common sense, eludes everyone at the NRA.
A few years ago, mayors Michael Bloomberg of New York and Thomas Menino of Boston organised a group of mayors to speak out against gun violence, on the grounds that neither the White House nor Congress appeared willing to do so.. They were not popular with the NRA, and their movement went quiet. Now, mayor Bloomberg has again urged the President to crack down on guns. But the bald and distressing fact is that all too many Americans are intoxicated with the glamour of guns and indifferent to the havoc and horror they create. Worse, they regard them as fuel for the kind of anarchic, nihilist entertainment in which they rejoice.
Such appetites may, and should, be easily satisfied with gun clubs, where gun nuts can shoot at inanimate targets to their hearts' content. Why don't they? Because such a skilful sport lacks the one ingredient which infatuates them the most - the risk of death through the exercise of individual power.
Of course, there is no reason on earth why any American should heed what I say, and they might even consider my words an impertinence. Indeed, in a lifetime of writing about crime and criminals, I have never argued anything with so little expectation of success. But what really depresses me is that even if America's police chiefs, their parsons, their heroes, or their President, were to say something similar, they would pay no attention at all.
Brian Masters is the author of The Evil That Men Do and Killing for Company: The Case of Dennis Nilsen.
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May 2, 2016
Move over Sarah Palin and Barack Obama, eastern Carolina Congressman Walter B. Jones is now stealing the spotlight on Saturday Night Live
Jones was featured in a parody on the show this past Saturday.
We asked the congressman about the skit-- that he just happened to watch live at his home in Farmville when it aired. Jones says, "So I thought this is funny this is cute satire of course then the next thing I see is Walter Jones, I was shocked, but I thought it was funny, very clever, very fair."
The parody was about the bailout of the automotive companies.
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When was the last time you checked out local.woot? Have you found any good deals there?
I feel sorry for @slydon, @unclepilot, and @inkycatz... look at all the questions they've asked!
10 Answers answer
I used it a month or so ago to get opinions on veterinarians as I was traveling to Seattle and my cat needed surgery. Not only was I given the name of a great doc, but I saved a huge amount of money!
I can only agree with @rprebel, but add 200 miles to it.
Closest city is a few hundred miles from me - if it had some closer deals, I would be all over it.
Local Woot is comparable to Apple Maps .... a failure.
I didn't even know they had cloned the deals format over to local. It's not really of use to me, either, as the closest is 500 miles away...sometimes it's a hassle to use groupings that are 20 miles away :/
Maybe I would look occasionally if it showed up on the bar at the top, but it seems like the red-headed stepchild of the woot family.
Oh wow, I completely forgot about it, especially since there were no deals close-by.
I checked it about a month ago, before I was headed to Seattle for the weekend. None of the current deals were anything useful for me.
Maybe a month ago
Another vote for never, except I'm like 1200 miles away from the closest one.
None of the local.woots are local to me, so I never go there. I believe the closest one is /dallas, and that's 200 miles away.


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|September 2003
Market Your Accessibility
Without exaggeration, you can probably say that everyone would enjoy and benefit from visiting
your science center or cultural institution. However, for a variety of reasons, people with
disabilities may not be coming. How can this be? After all, anyone may have a disability no
matter their age, gender, or background. In fact, one in every six people in the U.S. has a disability.
That's a sizeable number of people (54 million) with an estimated disposable income of 214 billion dollars.
Are people with disabilities waiting to be asked? What actions can you take to insure that they will be welcomed?
Spend smarter, not more. Begin
by taking an inventory of how you currently market your institution's
accessibility, and then consider what new marketing strategies you
might employ.
Work with your visitor services and floor staff to make a list
of what accessible services are currently in place. For many
science centers and museums, the list will include assistive listening
devices for lectures and large-format films; audio-described tours
and films; sign language interpretation; wheelchairs or scooters
on loan; brochures in large print and in Braille; tactile maps of
permanent exhibitions; newsletter on disk or audio cassette.
Gather examples of all the print materials you currently employ to market your institution to the general public. Newsletters, brochures, maps, annual reports, school and camp programs will likely be among these pieces. Next, make a list of other ways your institution markets its programs, exhibits, and services. On your list may be radio and television public announcements, mailings, telemarketing, and web pages. Get print copies of these messages so you can make a complete review of your current marketing tools.
Make accessibility explicit. Knowing what services are
available and how to request them allows visitors to plan ahead.
To what extent do your museum's various communication pieces address
accessibility? If the answer is "not much," don't be flustered,
start with what you can assure and clearly state these services
in all your brochures, on your web site, in your newsletter, and
in your press releases.
|Respect the power of language.
In addition to using disability access symbols in the above statements,
did you notice the language? You won't find the word "special." Also,
note the use of "people-first language." That's putting the person
first as in person who is blind, person who is hard of hearing, person
who uses a wheelchair. The language you use makes a powerful statement
about the credibility of your institution.
|Connect with this new audience.
While individuals with disabilities get their news about local programs and events just as the majority of the population does, many also rely on newsletters, listservs, web pages, and public service announcements produced and managed by local disability-related groups, organizations, and agencies. What they say matters.
Become familiar with these groups and how they communicate places to visit and things to do:
| When
you speak with a group or organization explain that your science
center or museum wants to make sure people with disabilities know
about its programs, events, and new films and exhibition, and you
need their help to do this. Would they be willing to include information
through their various communication outlets? If the answer is yes,
find out the name of the person you should contact. You will want
to talk with him/her about text length, deadlines, and the preferred
format for receiving your information (e.g., via e-mail, fax, or mail).
|Follow-up by sending a note to the people you spoke with, thanking them for their cooperation and reiterating your hope that they will keep you informed. But don't wait for them to call you; call them back in three months for feedback.
|Don't forget older adults and college students. Older adults are a large and growing demographic group in the U.S. while students with disabilities attending local colleges and universities are a renewable audience source. Both groups represent audiences for your programs and candidates for your volunteer corps.
While you can connect directly with students through a college's
office for students with disabilities, older individuals may or
may not self-identify as having a disability and likely rely on
many sources for their news and information. Among the news sources
older adults may use are local agencies and groups who work for
and with older adults. You will want to contact these organizations
and, if they agree, add them to your list of groups to receive announcements
on a regular basis.
|Achieve success quickly. There is no way to measure the effect of word-of-mouth publicity, but you can be reassured that "grapevines" exist. Similarly, within every community there are individuals who can act as advocates and spokespersons on your behalf. Know who these influential community members are.
Invite them to events; provide them with free passes; make sure they know you need and welcome their counsel.
your staff and volunteers. Getting visitors with disabilities
to come is one thing; their having a great time, is another. Good
experiences depend in large part on visitors' interactions with staff
and volunteers. Things fall apart when staff and volunteers do not
know what services are available, how to respond to questions and
complaints, or who on staff is responsible for accessibility services.
New, as well as experienced staff and volunteers need up-to-date information,
reminders, practice, and backup if they are to give quality customer
|Identify any remaining physical barriers and make a plan to remove them.
It's not unusual that your institution may still need to remove specific physical barriers so that visitors with
disabilities can fully participate in your events and programs. Rather than assuming that there are no barriers,
find out. Take these steps:
The most efficient transition-implementation plans include what needs to be done, who is responsible, the expected date of completion, and budgetary implications. Some changes can be accomplished with little or no money while others will take more time and resources.
With a plan, you can respond to questions and even complaints with "We appreciate your feedback. We know it's a problem, and we are working on it. We expect the work to be completed by ____. We value you as a visitor."
any missing accessible services. Removing physical barriers is
important; adding accessibility services is, too. These services may
mean the difference between individuals with disabilities visiting
or not.
Ask yourself: Do we know how to find sign language interpreters? Are our films and videos captioned? Which assistive listening systems would work best in our theater and for our films, lectures, meetings, and tours? Who can Braille print materials for us? Is the map of our floor plan available in large print? Do we have a tactile version? Are brochures and newsletters available on audio cassette or disk?
Your colleagues at other cultural institutions may have answers for you, and
the disability-related agencies and organizations in your community
can point you to helpful sources as well.
|Feedback is important to know how well your marketing is working. There may be no need to change how you hear from visitors, but you will want to check on how inclusive the processes are.
Take comments cards. Ask yourself: Are some available in 18 point type sans serif? Are cards within reach?
|It takes time to build an audience.
People with disabilities have a variety of interests as well as experiences with cultural institutions. In fact, you may run up against the perception "there is nothing there for us." Fortunately, perceptions can change. Change, however, depends in large part on the leadership of the marketing department. Continue to listen to people with disabilities and the organizations that represent them and your audience will grow, and your institution will prosper.
Others' Experiences for more ideas and please
Share Your Own.
      For more information
|Accessible Practices EXCHANGE is supported by the National Science Foundation under Grants No. ESI-9814917 and HRD 9906095. Opinions or recommendations expressed in this material are those of the author and presenters and not necessarily those of the National Science Foundation. www.nsf.gov
|ASTC is not responsible for the enforcement of the Americans with Disabilities Act (ADA) or Section 504 of the Rehabilitation Act of 1973. The information presented here is intended solely as informal guidance, and is neither a determination of your legal rights or responsibilities under the ADA, nor binding on any agency with enforcement responsibility under the ADA. This web site is not intended to offer legal, architectural, engineering, or similar professional advice. You should refer specific questions to an attorney, and/or national, state, and local ADA authorities.
|Copyright 2006 by the Association of Science-Technology Centers Incorporated. All rights reserved.


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Channing Tatum Opens The Book Of Life
Zoe Saldana, Diego Luna and more aboard
Usually when we learn about animated films in the works, there are still a couple of years left until they hit our screens. But with Reel FX's The Book Of Life, we can expect the 'toon next year. Channing Tatum, Zoe Saldana, Diego Luna, Ice Cube and Ron Perlman are among those who are providing their tonsil talents.
Co-writer and director Jorge Gutierrez dreamed up the idea, which The Hollywood Reporter is describing as the journey of Manolo, a young man torn between living up to his family's expectations and listening to his heart.
Before he makes his final decision, he sets of on an adventure across three fantastical worlds, and must also face his greatest fears. With plans to present a fresh visual style and new takes on popular pop songs (not that unusual for an animated movie, we suppose), the cast also features Cheech Marin, Hector Elizondo, Placido Domingo, Ana de la Reguera and Danny Trejo.
Guillermo del Toro, because he clearly doesn't have enough projects to oversee already, is on board to produce. 20th Century Fox Animation is set to release the film – in the US at least – on October 7, 2014.


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The Minister for Information, Youth, Culture and Sports, Fenella Mukangara, yesterday launched the Uhuru Marathon to be held in Dar es Salaam tomorrow.
She said the Government is committed to see an increased number of international marathon events being staged to attract foreign competitors.
The Uhuru Marathon is organised by Intellectuals Communication Company aiming at promoting unity and peace in the country while marking the Independence Day to be celebrated on Sunday.
The minister said it's a good development that the country is staging many marathon events.
She thanked the company for organizing the marathon which is staged in concurrence with the Independence Day celebrations.
She assured the organizers of the event that the Government will support them to achieve intended goals and also create a conducive environment so that people of all walks of life could take part at the event.
Speaking earlier, organizing firm's director Innocent Melleck said the marathon also aims at promoting athletics.
Most of the domestic based and international athletes are expected to compete.
The organizing firm donated T-shirts to President Jakaya Kikwete and Speaker of the National Assembly Anne Makinda as a gesture for their contribution to athletics development in the country. Melleck asked the minister to convey a message to the President so that he could be the Patron of the Uhuru Marathon.


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ACCA F2 Management Accounting
by Know Your Subject Limited
Developer Notes
Revision Software for ACCA F2 (2016 Exam Syllabus).
To develop knowledge and understanding of how to prepare and process basic cost and quantitative information to support management in planning and decision-making in a variety of business contexts.[ACCA]
This module has over 370 questions and...


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I have not been to a game in so long I barely remember how it's played. Weeks. It's been so long I'm actually craving a hot dog.
Look how lovely is the dog in the picture. I made that, not the fancy container or the onions or the sauerkraut, but the mustard. Well, I applied the mustard.
The Rangers are in town next week. I was supposed to be going Tuesday and Thursday. Now it seems I'm going Thursday only. Day Game! I think we have 6 or 8 tickets between all of us. Maybe that's a good time for my next Peanut Competition (Here are the Results of a previous competition). Always good to have many hands and mouths when you're comparing salty snacks. I'm going to work on that.


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Gov. Bobby Jindal's administration says it hasn't included money for state employee pay raises in its 2010-11 budget request to the Legislature. But that doesn't mean workers will go without a raise next year. Deputy Commissioner of Administration Barbara Goodson told the House Appropriations Committee last week that state agencies still could decide on their own to grant raises if they can carve out savings elsewhere in their budgets. "The only way we can do it is if we find the money," Goodson said. That answer did not sit well with some on the committee, who have been trying to curb the near-automatic "merit raises" granted to state workers even as state revenues are flat. "Each line item (in the budget) has some fluff in it," said Rep. John Schroder, R-Covington. "We fluff ourselves to death in this place."
State agencies must find the money if they want to give raises
Times-Picayune Staff By
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on March 07, 2010 at 6:15 AM
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on March 07, 2010 at 6:15 AM


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David Brent "Cheese" McWilliams, 70, passed away after a short-term battle with lung cancer on November 9, 2013, at his home in Orlando, Florida.
He is survived by his wife, Brenda Kay (Cox); daughters, Kimberly McWilliams of Florida and Kelly Kay Sipes (son-in-law Andy Sipes) of Heltonville, IN; and sons, Kevin Brent McWilliams (daughter-in-law Shelly Sowders McWilliams) of Noblesville, IN, and Kip David McWilliams (daughter-in-law Melissa Fadley McWilliams) of Ft. Wayne, IN. He was the grandfather of three wonderful children, Curtiss Lee Malicoat Jr., Jamie Kay Sipes, and Jesse Brent Sipes, along with his first great-grandchild, Lexie Kay, to arrive this month.
He was preceded in death by his parents, Iva T. King (stepfather James King) and Everett David McWilliams (stepmother Thelma McWilliams), and also a sister, Betty Lou Warner.
Born and raised in Columbus, Indiana, he was a graduate of Columbus North High School in 1961. He was an auto parts businessman working at "Robertson Auto Parts" in Columbus, then moving to Bloomington in 1965, owning his own business at "Big Red Auto Parts" and later working at "Big A Auto Parts." He moved to Florida in 1995 where he held the position of security guard at Walt Disney World in Orlando, retiring in 2005.
A memorial service will be held, following his cremation, on Saturday, November 23, 2013, at 1:00 p.m. at the Deremiah-Frye Mortuary Greene & Harrell Chapel, 700 W. Gourley Pike, Bloomington, IN 47404. A visitation will be held preceding the service from 11:00 a.m. to 1:00 p.m.
Memorial donations may be made in David's name to Cornerstone Hospice, 8009 South Orange Avenue, Orlando, FL 32803, phone number 1-800-679-6088.
Online condolences may be expressed to the family at www.DeremiahFryeMortuary.com.
All content copyright ©2016 The Republic, a publication of AIM Media Indiana unless otherwise noted.
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About Rimt Institute Of Engineering & Technology
Rimt Institute Of Engineering And Technology is affiliated to Punjab Technical University. The College offers Bachelor of Technology and Master of Technology courses. RIMT- the hub of knowledge is rising towards greater heights with every dawn. Under the aegis of Shri Om Parkash Bansal Educational and Social Welfare Trust, Mandi Gobindgarh and able stewardship of Chairman Shri Hukam Chand Bansal RIMT has been providing seamless and unlimited growth to its students.


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Newport, Oregon Bed and BreakfastsHome > Find a B&B > By City > Newport, Oregon
No trip to the Oregon Coast is complete without a visit to Newport, home to some of Oregon's unique attractions, and most beautiful scenery.
In South Beach, the Oregon Coast Aquarium features sharks, turtles and exotic fish. Rogue Brewery offers unique brews and daily tours. And there are always the old favorites, Wax Works, and Ripley's Believe it or Not. Newport also has a nice variety of restaurants, and a nice selection of inns and bed and breakfasts to meet your lodging needs.
Just out of town, be sure to visit Newport's two historic lighthouses, estuaries and forest trails. Whether for birdwatchers, walkers, joggers or bikers, Newport is the perfect recreation destination.Downtown | 1 Mile | 5 Miles | 10 Miles | 15 Miles | 25 Miles | 50 Miles
Click a zoom level above to see inns within a radius from the center of town(the red dot). Click a house icon to view more information about that inn.
The Lake House Bed and BreakfastPhone: 541-996-8938
Toll Free: 888-996-8938
Our lovely 2700 sq. ft. home with two suites is located on a 680-acre freshwater lake just 2 miles from downtown Licoln City, which offers 7 miles of beaches to experience the sounds and sights of the great Pacific Ocean.Lincoln City, Oregon
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Brey House Oceanview B&BPhone: 541-994-7123
The ocean awaits you just across the street.Private baths, private entrances.Wonderful breakfast served in the ocean view breakfast room.Quilts, snuggy warm blankets, fluffy bath towels.Fireplaces, tv, DVD, small in -room refrigatorsLincoln City, Oregon
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Blue Heron InnPhone: 541-997-4091
Toll Free: 1-800-997-7780
This two story 1940's ranch style home, with nooks and crannies, has been renovated with modern comforts, jetted tubs, and antiques. Enjoy the beautiful view over the Siuslaw River. Historic Old Town Florence, the Oregon Dunes, Heceta Lighthouse, and the convention center are just minutes away.Florence, Oregon
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Grand Victorian B&B and Vintage Cottage RentalPhone: 541-265-4490
Toll Free: 800-784-9936
Welcome to our lovely Victorian oceanview B & B. Schedule your romantic wedding, elopement, anniversary, birthday or getaway with us. Guests love the appetizers, desserts and big hot breakfasts (optional seating times). The oceanview Vintage Cottage is perfect for couples or for a family. Enjoy!!!Newport, Oregon
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Tyee Lodge Oceanfront B&BPhone: 541-265-8953
Toll Free: 1-888-553-8933
Tyee Lodge offers gracious Oregon Coast lodging for travelers in a picturesque beachfront setting. Spectacular views of the Pacific Ocean and the incomparable Oregon coastline can be seen from every window. All rooms have private baths. Close to Newport, Oregon and other Central Oregon Coast attractions including the Bayfront, historic Nye Beach, ocean tour cruises and fishing, two lighthouses, the Oregon Coast Aquarium, and fantastic restaurants. We are here to help you discover why guests return again and again to Tyee Lodge. Our spacious themed rooms have queen size beds with cozy chairs for reading, whale watching, or enjoying a storm. Outside decks are made for catching fresh sea air and relaxing views of the mighty Pacific and shore pines. It's the perfect spot for bird watching, including a resident Eagle. A short, gently sloping private path leads to miles of incomparable Oregon beach.Newport, Oregon
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Ocean House Bed and BreakfastPhone: 541-265-3888
A couple's retreat at the BeachNewport, Oregon
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NEW YORK ( TheStreet) -- When a smaller company takes over a larger company the synergies can be exponential.That's exactly what I'm anticipating when IntercontinentalExchange (ICE - Get Report) completes its acquisition of NYSE Euronext (NYX) sometime in the second half of this year.
Euronext is the European electronic stock-exchange business that operates markets in Paris, Amsterdam, Brussels and Lisbon. The spinoff was outlined as part of the $8.2 billion transaction announced last month between ICE and NYX. The spinoff has an important goal. It will, according to the newspaper, help to "...allay potential concerns that exchanges managed from afar could diminish the influence of European financial hubs, hurting their ability to control jobs and other aspects of the business." During its conference call, NYX reiterated something first mentioned in a Jan. 31 Wall Street Journal article, that a side deal NYX reached with ICE could prevent a competing bid from arising. Under the deal, NYSE's European derivatives unit, known as Liffe, agreed to clear its trades through IntercontinentalExchange for at least two years, the newspaper reported, citing unnamed sources. The deal will remain valid regardless of whether the merger with IntercontinentalExchange closes.


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The Federal Utility Partnership Working Group (FUPWG) establishes partnerships and facilitates communications among Federal agencies, utilities, and energy service companies. The group develops strategies to implement cost-effective energy efficiency and water conservation projects through utility incentive programs at Federal sites.
The mission and objectives of the Federal Utility Partnership Working Group are to:
Enhance existing or foster new partnerships between Federal agencies and their servicing utilities to identify, develop, and implement cost-effective energy efficiency, water conservation, and renewable energy projects at Federal sites
Identify how utilities can help Federal agencies meet energy management goals required by legislation
Expand the communication infrastructure between Federal agencies, specific sites, and utilities regarding all aspects of Federal energy management
Prepare agencies for upcoming changes in the electric utility industry and provide information regarding recent technological developments in the areas of energy efficiency and water conservation.
Read about the FUPWG's purpose, policies, and operations.
FUPWG participants include Federal agencies, utilities, and energy service organizations. See a list of FUPWG participants.
The Federal Utility Partnership Working Group meets twice a year to share success stories, information on FEMP programs and products, and other FUPWG business. Past meeting summaries are available. For upcoming meetings, please see the FEMP calendar of events.
To submit utility energy service contract (UESC) data to help the Federal Energy Management Program improve its UESC activities, fill out the following documents and send them to Kaila Raybuck. All information is kept confidential.
Contacts | Web Site Policies |
U.S. Department of Energy | USA.gov
Content Last Updated: 09/26/2013


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Sunday Nov 09 2008
By: Jim Ratajczak, The Telegraph
EL DORADO HILLS—The Vista del Lago varsity Eagles were dealt their seventh loss of their inaugural season Friday night, this one at the hands of the Union Mine Diamondbacks. But the game's final score, 27-7, doesn't tell the whole story. It was a game the Eagles could, and probably should, have won. "The first half of the game, we played extremely well," said Vista head coach Chris Jones. "But we fizzled out." Union Mine had the game's first scoring opportunity with the ball on the Vista 10 but missed the field goal. Vista took this momentum and ran with it, as Eagles running back Trey Hairabedian, who led the offensive attack downfield, took the handoff from quarterback Anthony Mull in for a touchdown late in the first. But in the second quarter, linebacker Kody Kenworthy picked the first Mull's four interceptions at the Vista 49 yard line, scored on a handoff moments later and the teams went into the half tied at 7 apiece. With 9:47 left in the third, Kenworthy picked off Mull's pass once more and, with only Mull to beat, ran the ball into the end zone, giving the Diamondbacks a 14-7 lead. Neither team put points on the board for the rest of the third, thanks in part to stout defensive efforts and penalties. However, the game came to a screeching halt in the fourth when Vista defensive standout Nick Marshall was injured trying to keep Union Mine running back Mike Rigsby from reaching the end zone. Play was stopped for several minutes and a hush fell over the crowd as Marshall lay on the ground, flanked by trainers and coaches. Eventually, he was carried off the field in a stretcher by emergency medical technicians. When play resumed, Union Mine had a 20-7 lead. But it was obvious the Vista team had lost focus. "He's one of our main leaders on defense," said tight end Ricky Loewen of Marshall. "We were all worried about him. It looked like his neck was really injured." "That was hard," said Jones. "That takes a game and brings it to a screeching halt." Vista team officials said Marshall was responding well and that he was taken off the field as a precaution. The Diamondbacks scored for the final time on another Mull interception, this time by Rigsby. In many ways, it was a frustrating game for Vista. But Jones still hopes to build on the experience. "We did a lot of really good things," he said. "We talk about and try to emphasize doing a lot of things well." The Eagles will travel for their final game of the season Saturday against Christian Brothers. Although Vista's sole wins came in Saturday wins, Jones anticipated a week of intense practice. "We've talked to the kids about getting ready to compete against a team battling for a playoff spot," he said. JV at a glance The junior varsity Eagles suffered their fourth loss in five contests Friday night, dropping their game against Union Mine 35-6. The only points for the Eagles came late in the third when wide receiver David Slayton caught a touchdown pass from quarterback Henry Kozlowski.