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Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2 receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.
- lung
- mucus
- exocytosis
in healthy lungs, the viscoelastic mucus gel plays a major role in mucociliary clearance (29), a key component of host defense. Polymeric mucins, the principal component of mucus, are high-molecular-weight glycoconjugates secreted by airway goblet cells and submucosal glands. In airway obstructive diseases such as chronic bronchitis and cystic fibrosis, the airways can become occluded as a result of mucus hypersecretion caused by goblet cell and glandular hyper- or metaplasia, and early phases of these diseases implicate goblet cells in the lower airways selectively (23). While substantial knowledge exists with regard to the regulation of goblet cell secretion by agonists and intracellular messengers (10, 11, 28), very little is known about the downstream effectors activated after cell stimulation. For example, numerous studies have shown that goblet cells respond primarily to purinergic agonist stimulation via a luminal P2Y2 receptor (P2Y2R) that couples through Gq to phospholipase C (PLC) to generate diacylglycerol and inositol 1,4,5-trisphosphate, with a resulting activation of PKC and mobilization of Ca2+ (12, 28). The downstream effector molecules activated by PKC and Ca2+ that direct and regulate mucin granule exocytosis, however, are virtually unknown.
Actin is a structural protein of great importance in eukaryotic cells, playing a major role in muscle contraction, cell motility, and many other cell functions. During the past 20 years, actin filaments have also been shown to regulate membrane trafficking, especially as it relates to endo- and exocytosis (for review, see Refs. 14, 16). Classically, cortical actin filaments are hypothesized to negatively regulate endocrine secretory cell exocytosis by forming a physical barrier between secretory granules and docking sites; in this article, we refer to this hypothesis as "the barrier hypothesis." In chromaffin cells, for example, several studies have demonstrated that the disruption of actin filaments is essential for exocytosis (18, 59). Disruption of the cortical actin barrier before exocytosis is hypothesized to be caused by two proteins acting in parallel (60). First, the PKC-activated protein myristoylated alanine-rich C kinase substrate (MARCKS; see Ref. 4) tethers actin filaments to the plasma membrane under resting conditions and translocates to the cytosol upon phosphorylation by PKC, thereby disrupting the cytoskeleton. In chromaffin cells (49), platelets (15), and bovine luteal cells (52), the phosphorylation of the membrane-bound MARCKS protein is required for regulated exocytosis. Second, scinderin, a Ca2+-activated, gelsolin-related actin filament-severing and -capping protein (see Refs. 34, 48) has been implicated in cytoskeletal disruption occurring before exocytosis. In stimulated chromaffin cells, scinderin has been shown to control the disassembly of the cortical F-actin network after activation by Ca2+ (59, 62).
Alternatively, more recent data suggest that actin filaments act positively in regulated exocytosis, presumably by transporting or positioning secretory granules relative to plasma membrane docking sites (see, e.g., Refs. 17, 41).
The role of actin filaments in airway goblet cell secretion is generally unknown. In the present study, we investigated the role of actin filaments, MARCKS, and scinderin in regulated mucin secretion, primarily by using SPOC1 cells, a cell line derived from rat trachea that secretes mucin in response to stimulation by P2Y2R agonists (ATP, UTP, and ATPγS), phorbol 12-myristate 13-acetate (PMA), or Ca2+ (2).
DMEM-F-12 culture medium was obtained from GIBCO-BRL (Gaithersburg, MD), and the supplements were purchased from Collaborative Research (Bedford, MA). The following biochemicals were purchased from the following commercial sources: PMA and jasplakinolide, Calbiochem (La Jolla, CA); ATPγS, Roche Applied Science (Indianapolis, IN); ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), Sigma (St. Louis, MO); latrunculin A, Molecular Probes (Eugene, OR); and streptolysin O (SLO), Corgenix (Peterborough, UK). Synthetic peptides representing the actin-binding domains of scinderin and their scrambled controls were synthesized in the University of North Carolina Peptide Core Facility and had the following amino acid sequences (after 66): Sc-ABP1, AAIFTVQMDDYL; scrambled1, YAALMFDIDATQV; Sc-ABP2, RLLHVKGRR; and scrambled2, LVRGKRRLH.
The following antibodies were purchased or received from the following sources: pan-actin monoclonal antibody (Ab-1), Oncogene (Boston, MA); β-actin-specific monoclonal antibody (AC15), Sigma; γ-actin-specific polyclonal antibody (43), J. C. Bulinski (Dept. of Biological Sciences, Columbia University, New York, NY); green fluorescent protein (GFP) polyclonal antibody, Abcam (Cambridge, UK); MUC5AC-specific polyclonal antibody (54), J. K. Sheehan (Dept. of Biochemistry & Biophysics, University of North Carolina, Chapel Hill, NC); phospho-MARCKS-specific polyclonal antibody (sc-12971-R), Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal anti-scinderin antibody was raised in rat using recombinant mouse scinderin as the immunogen (Laboratory of Hormonology, Marloie, Belgium). The antibody was purified by affinity chromatography over a cyanogen bromide-Sepharose mouse scinderin column, and its specificity was confirmed using ELISA and Western blotting. The anti-scinderin antibody recognized 50 ng of recombinant mouse scinderin and stained only very weakly for 1 μg of human plasma gelsolin.
Secondary antibodies conjugated with either Alexa Fluor 488 or Alexa Fluor 594 were purchased from Molecular Probes for immunostaining. Secondary antibodies for Western blotting were purchased form Jackson ImmunoResearch (West Grove, PA).
For control Western blot analysis, whole cell extracts of human bronchial epithelial cell primary cultures and purified polymorphoneutrophils and red blood cell ghosts were obtained from Scott H. Randell and Hirotoshi Matsui (University of North Carolina, Chapel Hill, NC), respectively, and Peter Mohler and Vann Bennett (Duke University, Durham, NC).
SPOC1 cell culture, mucin secretion experiments, and tracheal xenografts.
SPOC1 cells were seeded as appropriate to the experiment in 6-, 12-, or 48-well cluster plates (Costar, Cambridge, MA) at 8,850 cells/cm2 and were maintained in rat tracheal epithelial cell medium (47) as previously described (3, 50). Except for cells grown solely for passaging, the medium also contained 10 nM retinoic acid. Culture media were changed daily, and the cultures were used for experiments after differentiation of a mucin-secreting phenotype 14–21 days postconfluence. Mature SPOC1 cells were used for mucin secretion experiments after being washed gently three times with warmed DMEM-F-12 separated by 30 min each. The experimental media were then added, and the cells were incubated as indicated in results, after which the media were harvested for mucin assays. For the experiment shown in Fig. 3, SPOC1 cells harvested after routine passaging were seeded into denuded, precannulated rat tracheas at ∼1 × 106 cells/graft in a volume of 50 μl. After the cannulas were heat sealed, the grafts were implanted subcutaneously into the backs of athymic nu/nu BALB/c mice and harvested after a 3-wk incubation using previously described procedures (8). At harvest, the xenografts were opened along the midlines, the mucus plug was gently washed away with DMEM-F-12, and the tissue was incubated for 20 min either without or with 100 μM ATPγS in DMEM-F-12. All animal procedures were performed according to University of North Carolina Institutional Animal Care and Use Committee-approved protocols.
SPOC1 cell permeabilization.
SPOC1 cells were permeabilized using SLO as described previously (50, 53). Briefly, the cells were washed with PBS and then with intracellular buffer (in mM: 130 K-glutamate, 20 PIPES, 1 MgATP and MgCl2, and 3 EGTA, pH 6.8). Free Ca2+ in intracellular buffer was set to 0.1 μM, except in solutions designed to stimulate mucin release, in which it was raised to 10 μM (50, 53). SPOC1 cells were permeabilized with SLO (1 U/ml in intracellular buffer) for 30 s, washed once in intracellular buffer, then incubated with the appropriate experimental solution for 15 min, and samples were collected for mucin analysis. TO-PRO (Molecular Probes, Eugene, OR), a membrane-impermeant, DNA-binding fluorescent dye, was used to confirm cell permeability as previously described (53).
Mucin enzyme-linked lectin assay.
Samples collected from experiments were assessed for mucin content using an enzyme-linked lectin assay described previously (3). Briefly, samples in 96-well high-binding microtiter plates (Costar; Corning Life Sciences, Acton, MA) were incubated overnight at 4°C or 37°C for 2 h, washed with PBS-0.05% Tween 20, and incubated with 2.5 μg/ml of horseradish peroxidase-labeled soybean agglutinin lectin for 1 h at 37°C. The plates were washed, developed with O-phenylenediamine in a citrate phosphate buffer (0.0175 M, 0.01% hydrogen peroxide, pH 5.0), stopped with 4 M H2SO4, and analyzed for optical density at 490 nm in a microtiter plate reader (Dynatech, Alexandria, VA). Standard curves were generated from purified SPOC1 mucins applied to each plate, and the results were expressed in nanograms of mucin released per culture. Importantly, in Western blotting of SPOC1 cell extracts, soybean agglutinin stains only high-molecular-weight glycoconjugates (3).
Western blotting.
After washing and equilibration, SPOC1 cultures were incubated as stated in results, and the cells were washed with ice-cold PBS and then lysed into one of three ice-cold lysis buffers (1): whole cell lysates, 20 mM Tris·HCl, pH 7.4, 2 mM EGTA and EDTA, 1% NP-40, and protease inhibitor cocktail (P-8340, 1:20 dilution; Sigma); cell fraction buffer: the same ingredients, but without NP-40; special lysis buffer for phospho-MARCKS (38) (in mM): 20 Tris·HCl, pH 7.4, 10 EGTA, 2 EDTA, 1 PMSF, 250 sucrose, 2 sodium vanadate, 25 sodium fluoride, 1% NP-40, and protease inhibitor cocktail (P-8340, 1:20 dilution; Sigma). For fractionation, lysed cells were homogenized on ice (15 strokes, Potter-Elvehjem tissue grinder), the extract was centrifuged for 1 h at 100,000 g, and the supernatant was taken as the cytosolic fraction. The pellet was solubilized in buffer supplemented with 1% NP-40, incubated 30–60 min, and centrifuged (14,000 rpm), and the supernatant was taken as the membrane fraction. Importantly, the membrane fraction also contained associated cytoskeleton.
Proteins in cell extracts or fractions were resolved by performing 10% SDS-PAGE and transferred electrophoretically to PVDF membranes. The blots were probed with antibodies appropriate to the experiment, and immunoreactive bands were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL). Imaging of the Western blots was performed with a platform scanner at a resolution of 1,200 × 1,200 dpi × 12 bits and quantitated using MetaMorph image-processing software (Universal Imaging, Downingtown, PA).
Immunolocalization and microscopy.
Paraffin sections (6 μm) of human lung fixed for 10 min in 10% neutral buffered formalin (NBF) were provided by the University of North Carolina Cystic Fibrosis Center histology facility. Cystic fibrosis (CF) and non-CF tissues were used. The tissues were procured from patients with CF and patients without lung disease in accordance with the guidelines of the Committee on the Protection of the Rights of Human Subjects of the University of North Carolina at Chapel Hill. SPOC1 cell xenografts were fixed in 10% NBF and processed for histology, and paraffin sections were cut at 6 μm. Sections were deparaffinized and rehydrated, blocked for 1 h in 3% BSA, and washed. Next, the first antibody was applied with an overnight incubation, followed by washes and a 1-h second antibody incubation. The slides were mounted with an antifade medium (Vectashield; Vector Laboratories, Burlingame, CA).
Immunostained xenograft sections were examined under a Leica DMIRB inverted microscope outfitted for epifluorescence and differential interference contrast using a PlanApo ×100/1.4 oil-immersion objective lens (Leica Microsystems, Wetzlar, Germany) and images were acquired with an Orca-II B water-cooled, charge-coupled device camera (Hamamatsu, Welwyn Garden City, UK). The sections were illuminated with a 100-W HBO mercury lamp and collected after being passed through the appropriate dichroic filter sets: 595DCLP for Alexa Fluor 594 and Q505LP for Alexa Fluor 488 images. Immunostained lung sections were examined with a Zeiss LSM 510-Meta laser scanning confocal microscope using an AchroPlan ×63/1.4 oil-immersion objective lens (Carl Zeiss, Oberkochen, Germany). Images (8-frame averages) were acquired in two channels using the same optical thickness setting for each channel. Alexa Fluor 488 and 594 were excited with, respectively, an Ar laser (488 nm) and a HeNe1 laser (543 nm), and emissions were separated using a 488-/543-nm dichroic filter and passed through a 545-nm beam splitter. Green fluorescence was collected with a 518/25 band-pass filter, and red fluorescence was gathered with a 585 low-pass filter. Lack of cross talk was verified by successively turning off the excitation source adjacent to each channel and confirmed by no visible signal. Photomultiplier tube gain and offset were adjusted to achieve an optimal contrast and avoid saturation in the region of interest.
Molecular cloning.
Total RNA was extracted from SPOC1 cell line and human bronchial epithelial primary cell cultures using the RNeasy extraction kit (catalog no. 74104; Qiagen) and stored at −80°C. SPOC1 cell reverse transcription was achieved with 2 μg of RNA using a first-strand cDNA synthesis kit (Invitrogen) by applying oligo(dT) for mRNA probing and SuperScript II reverse transcriptase for polymerization, stored at −20°C. cDNA (1 μl) was amplified by performing PCR in a 25-μl reaction volume containing 10 mM 2-deoxynucleotide 5′-triphosphate (0.5 μl) and Taq (1 μl). Rat scinderin primers were designed using the known mouse scinderin and gelsolin sequences to be specific in a region of low homology between the two proteins. The scinderin primers were 5′-AACAGTGGTAGGGTCCAGATT-3′ and 5′-AGTGATAGATGCCAGGTTCCTC-3′, with a PCR product of 360 bp. The gelsolin primers were 5′-GGTTCCAACAAGGTGCCCGTG-3′ and 5′-GGTGGCTCCAGAGCTGCTGGC-3′, with a PCR product of 330 bp. The PCR program was predenaturation at 94°C for 3 min and 33 cycles of amplification at 94°C for 45 s, 62°C for 30 s, and 72°C for 45 s, followed by extension at 72°C for 10 min. PCR products were separated by performing electrophoresis in a 1.5% Tris-acetate-EDTA (TAE)-agarose gel run at 110 V for 30 min and visualized using ethidium bromide. Full-length scinderin clones were obtained by performing PCR using Pfu polymerase (GIBCO-BRL) using 5′-ATGGCGCAGGAGCTGCAGCACCCCGAGTT-3′ as forward primer and 5′-TTACCACCTGCTGGAGTCCCAGCCCAGAAA-3′ as reverse primer. The products were cloned using the ZeroBlunt TOPO PCR cloning kit (Invitrogen), and the nucleotide sequence of the products was determined by performing DNA sequencing. Human scinderin was cloned beginning with a blast search, using the mouse scinderin cDNA (GenBank accession no. U04354) that returned the human p-aminoclonidine sequence (GenBank accession no. AC005281). This sequence proved to encode an open reading frame of 1,602 bp with 89% homology to the coding region of the bovine scinderin cDNA (GenBank accession no. D26549) that contained the 5′ end of the gene. The remaining 3′ sequence was isolated by performing 3′ rapid amplification of cDNA ends (RACE) using the commercially available human brain Marathon Ready cDNA library (Clontech, Oxford, UK) according to the manufacturers instructions. Analysis of the RACE products by performing DNA sequencing (ABI Prism 310; PerkinElmer, Wokingham, UK) revealed a further 546 bp, completing the scinderin coding region. A full-length human scinderin cDNA was subsequently isolated from human brain Marathon Ready cDNA using PCR amplification with the gene-specific forward primer 5′-ATGGCGCGGGAGCTATACCACGAAGAGTTC-3′ and reverse primer 5′-TTACCACTTGCTGGAATCCCAGCCCAGGAA-3′. PCR products were obtained and cloned into the TA vector (Invitrogen, Paisley, UK), and DNA sequencing confirmed the full-length coding sequence to be 2,148 bp.
β- and γ-actin overexpression.
The retroviral expression plasmid pLXSNb was derived from the Moloney murine leukemia virus (42) with a long-terminal repeat (LTR) promoter to drive expression. This vector contains a cloning site and uses an internal SV40 promoter to regulate the neomycin resistance (NeoR) gene. To improve selection efficiency, the SV40 promoter was removed and replaced with a poliovirus internal ribosomal entry site (IRES). To obtain a higher level of expression, a second vector, QXCIN (Clontech), was used. This vector uses an internal cytomegalovirus (CMV) promoter to drive expression of the insert/IRES/NeoR cassette. The cDNA encoding for yellow fluorescent protein (YFP)-β-actin (pEYFP-actin, catalog no. 6902-1; Clontech) was inserted into the multiple cloning site for both vectors using NotI and BamHI digestion. Because γ-actin differs from the β-isoform by only four highly conserved amino acid residue substitutions at the NH2 terminus (as detailed in results), we generated YFP-γ-actin constructs using a four-point site-directed mutagenesis scheme within the YFP-β-actin sequence (QuickChange kit, catalog no. 200519-5; Stratagene). The mutagenic primers were 5′-CGAGCTATGGAAGAAGAAATGGCCGCGCTCGTCATCGAC-3′ and its complement, with the mutated sites underlined.
Retroviral particles were obtained by transfection of human embryonic kidney-293T packaging cells using a Ca2+-phosphate precipitation method (25); titers were determined to be between 5 × 105 and 5 × 106 viral cells/ml. Subsequently, SPOC1 cells in passage 6 were exposed to 1 ml of virus-containing solution for 4 h in the presence of 8 μg/ml of polybrene (Aldrich Chemical). The selection for gene transfer started the next day and continued for ∼1 wk with medium changed daily using G418 at a concentration of 125 μg/ml. Actin-overexpressing cells were used for experiments between passages 8 and 13.
Statistical analysis.
Data are presented as means ± SE for a specified number of SPOC1 cell cultures, with each culture in a given experiment originating from a different passage. ANOVA and Student's t-test were used as appropriate to determine statistical significance between data sets. Data were deemed statistically significant at P < 0.05.
Localization of actin filaments in polarized human airway epithelial cells.
To determine whether actin is positioned in well-differentiated goblet cells to play a role in exocytosis, human lung sections were immunostained and observed using confocal microscopy. For these experiments, CF tissues were used because of their higher incidence of goblet cells; however, lung tissues from patients free of lung disease were also stained as controls, and their more sporadic goblet cells showed similar staining patterns (data not shown). Paraffin sections were stained with a monoclonal pan-actin antibody and, to label goblet cells in the superficial epithelium, a polyclonal MUC5AC antibody (54). The luminal margin of the airway epithelium exhibited a distinctly apical distribution of actin, but one that was specific to cell type (Fig. 1). In ciliated cells, actin localized to the apical membrane and microvilli, which gave it a "bushy" appearance. In goblet cells, however, actin took on the appearance of a prominent subplasmalemmal sheet lying between the apical membrane and mucin granules. In both cell types, actin signals were weak in the cytoplasm and along the basolateral surface. Notably, actin was not detected within the secretory granule region of goblet cells. Stress fibers in the basal pole of the cells were not observed because of the orientation of the sections and our focus on the luminal aspect of the airways. Interestingly, actin in basal cells was distributed in the cytoplasm as well as in the cell cortex (data not shown).
Studies of nonpolarized, nonsecretory cells have shown that β- and γ-actin have different cellular distributions, with γ-actin present in stress fibers and in the cell cortex and β-actin localized to the cortex and in other specialized compartments (see Ref. 27). In epithelial cells, β-actin has been reported to localize preferentially to the apical membrane, whereas γ-actin associates with both apical and basolateral membranes (64, 65). We used actin isoform-specific antibodies to determine the distribution of these isoforms in airway epithelial cells. Figure 2 shows that β- and γ-actin have similar distributions in both ciliated and goblet cells, with both isoforms localizing uniformly to the apical membrane. As a control for specificity of the antibodies, note the bright neutrophil infiltrating the epithelium that stained strongly for β-actin but faintly positive for γ-actin (Fig. 2, asterisks). This difference in expression was confirmed by performing Western blotting on isolated neutrophil and red blood cell ghost extracts, which showed a signal 3.7 times stronger for β-actin than for γ-actin (Fig. 2).
Distribution of actin isoforms in SPOC1 cells before and after stimulation.
SPOC1 cells adopt a multilayered morphology when grown in culture, with the mucin-containing cells in the top layer being rather flat. To gain a columnar phenotype favorable for immunolocalization, we instead grew cells in denuded rat tracheas as xenografts in nude mice. Under these conditions, as shown in Fig. 3, A and B, SPOC1 cells grew as a pseudostratified epithelium in which all of the columnar cells had a goblet cell-like phenotype (see also Refs. 3, 47). In these cells, β- and γ-actin assumed the same cellular distribution observed in human airway goblet cells (Fig. 3B, left; compare with Fig. 2). To test whether apical actin filaments are altered in their distribution during regulated mucin secretion, we stained SPOC1 cell xenografts after 20 min of maximal stimulation by ATPγS. As shown in Fig. 3B, both β- and γ-actin virtually disappeared from the apical pole, a period during which the mucin pools in the cells were largely depleted. These results suggest that the actin filaments function as a barrier to secretion in goblet cells under resting conditions and show that they undergo dramatic rearrangement and disruption during purinergic stimulation.
Actin dynamics in SPOC1 cells: involvement of MARCKS and scinderin.
Because MARCKS and scinderin are hypothesized to effect actin filament disruption before exocytosis in other secretory cells (60), we tested whether these proteins are expressed in SPOC1 cells and/or in human airways and whether they might also participate in regulated mucin secretion. MARCKS expression in SPOC1 cells was tested by performing Western blotting of whole cell extracts obtained from cultures incubated for 40 min with DMEM-F-12 vehicle, a maximal dose of ATPγS (100 μM), or a dose of PMA (30 nM) sufficient to fully activate PKC (1, 3). The blots were probed with a phospho-MARCKS-specific antibody as an index of MARCKS translocation to the cytosol (38). The immunoblots (Fig. 4) indicate that MARCKS was phosphorylated progressively after stimulation by both ATPγS and PMA, reaching a peak at 10–20 min and declining slightly to a stable plateau. Hence, MARCKS protein appears to be expressed in SPOC1 cells and is phosphorylated after agonist stimulation, indicating a translocation from the membrane in a manner consistent with its postulated role in regulated secretion (60).
Human airway epithelial and SPOC1 cells were found to express scinderin mRNA by performing RT-PCR (Fig. 5A, left). Interestingly, gelsolin mRNA was expressed in human bronchial epithelial cells, but apparently not in SPOC1 cells, a result consistent with the notion that gelsolin and scinderin have mutually exclusive expression patterns in different cell types (33). The scinderin cDNAs were cloned and sequenced [GenBank accession nos. AF276507 (human) and NM_198748 (rat)], and an alignment of the bovine, rat, mouse, and human nucleotides sequences showed an identity of 79.3%. A scinderin-specific antibody used to probe a Western blot of SPOC1 cell extract showed that the protein was also expressed (Fig. 5A, right). In further analysis, scinderin was found in both the cytosol and membrane fractions, and its distribution did not appear to change after exposure of SPOC1 cells to purinergic agonist (data not shown).
To test whether scinderin possesses the capacity to sever actin filaments relevant to mucin granule exocytosis, synthetic peptides corresponding to the two actin-binding domains of scinderin (66) were presented to SLO-permeabilized SPOC1 cells activated to release mucin by elevating Ca2+ to 10 μM (53, 50). As shown in Fig. 5C, the presence of these peptides inhibited Ca2+-activated mucin secretion in a concentration-dependent manner, with a maximum of −41.7 ± 11.1% at 30 μM. Scrambled peptides used as a control under the same conditions had no effect on secretion. We interpreted these results to indicate that the synthetic peptides competed with the action of the endogenous scinderin in activated SPOC1 cells.
Actin disruption and stabilization effects in SPOC1 cells.
Disruption of the actin cortical network by cytochalasin D or the sponge-derived G actin-sequestering agents latrunculins A and B (57b) has been shown to induce secretion in chromaffin and other cells (9, 14). Consistent with these findings, latrunculin A treatment of SPOC1 cells resulted in a concentration-dependent increase in mucin secretion under both basal (nonagonist control) and agonist-stimulated conditions, with maximal effects at 10 μM of 77 and 85%, respectively (Fig. 6). These results suggest that actin filament disruption in mucin-secreting cells activates granule release and that it does so in a manner that maintains the responsiveness of the cells to agonist.
Conversely, stabilization of actin filaments with phalloidin or jasplakinolide (6) has been shown to inhibit regulated exocytosis (41, 51). In SLO-permeabilized SPOC1 cells activated by an elevation of Ca2+ concentration to 10 μM, jasplakinolide inhibited mucin release in a concentration-dependent manner, with maximal effect at 10 μM of −51.4 ± 22.3% (Fig. 7). Together, the latrunculin A and jasplakinolide data indicate that actin filament disassembly in SPOC1 cells is not only permissive but also necessary to enable mucin granule exocytosis upon stimulation by agonist.
Overexpression β- and γ-actin in SPOC1 cells.
As an independent test of the barrier hypothesis regarding regulated mucin secretion, β- and γ-actin were overexpressed in SPOC1 cells. Because SPOC1 cells require 2–3 wk to differentiate, a stable expression system was necessary; therefore, we used a retroviral expression system, repeating infections on individual lots of passage 6 cells at least four times to ensure that the results obtained were not due to inopportune insertion of the provirus. The retroviruses generated expressed either YFP-β-actin (22) or YFP-γ-actin. Because nonmuscle β-actin differs from nonmuscle γ-actin by only four amino acid residues in the NH2-terminal segment, Asp2-Asp3-Asp4- … -Val10 and Glu2-Glu3-Glu4- … -Ile10, respectively (20, 38), γ-actin vectors were obtained by site-directed mutagenesis of the β-actin vectors. The expression of each isoform was driven by either the LTR or the CMV promoter, leading, respectively, to weak or strong expression in the resulting cell lines. As a control, SPOC1 cells were infected using an empty retroviral vector. After selection, infected SPOC1 cells were grown for 15–18 days and then were either assessed for actin isoform expression levels or challenged with secretagogues for 30 min, and then mucin release was quantified. Data from the retrovirally infected SPOC1 cells were normalized to basal mucin release determined for each infected cell type.
YFP-actin expression was determined by performing Western blotting (Fig. 8, A and B, left) using a GFP antibody for YFP-actin and β- or γ-actin-specific antibodies for endogenous actin. Quantification of the blots revealed that YFP-actin expression driven by CMV was >2.2 times stronger than the expression driven by LTR. Estimation of overexpressed YFP-actin relative to endogenous actin was not possible, because the conjugated forms of β- or γ-actin were recognized only partially by pan-, β-, and γ-actin antibodies, likely because of camouflage of the epitope by the YFP protein.
Strikingly, overexpression of YFP-β- and YFP-γ-actin led to a decrease in mucin secretion in an expression level-dependent manner for cells stimulated with either 100 μM ATPγS or 100 nM PMA. SPOC1 cells expressing YFP-β- or YFP-γ-actin behind the weak LTR promoter exhibited inhibition ranging from 41 to 52% of control; for cells expressing actin behind the stronger CMV promoter, the inhibition ranged from 67 to 79% (Fig. 8, A and B, right).
To determine whether YFP actin enhanced the normal barrier function of the apical actin filaments or whether the inhibition observed was an artifact of overexpression, we treated YFP-β- and YFP-γ-actin-overexpressing cells with latrunculin A. As shown in Fig. 9, mucin secreted in the latrunculin A-treated cells attained the same level for non-, weakly, and strongly YFP-actin-overexpressing cells. First, the results of this experiment imply that actin-overexpressing and wild-type SPOC1 cells contain the same total amount of mucin. Second, the results show that inhibition of mucin secretion by actin isoform overexpression is specific to actin filaments. In control (empty vector) cells, latrunculin A increased the response to ATPγS, similar to its effect in noninfected cells (Fig. 6). Notably, latrunculin A stimulated the same nominal degree of response from all infected cells, except for those strongly overexpressing YFP-γ-actin. In the case of YFP-γ-actin driven by the CMV promoter, there may have been some nonspecific effects; however, even in this case, breakdown of the actin filament barrier resulted in strong stimulation of mucin secretion. Collectively, these findings are novel and reveal that actin overexpression inhibits mucin secretion in a manner that can be overcome by an actin filament-disrupting agent, offering additional support for the barrier hypothesis.
Actin is a ubiquitous protein that in nonmuscle cells is responsible for functions ranging from controlling cell shape and motility to serving as a major, membrane-associated protein scaffold. β- and γ-actin, the two predominant isoforms expressed in nonmuscle cells, differ by only four amino acids at the NH2 terminus (for review, see Refs. 20, 27) and, despite this similarity, are differentially distributed in many cells. In motile cells, for instance, γ-actin is distributed throughout the cell and in stress fibers, whereas β-actin localizes primarily to cortex, especially the leading edge (21, 24). In neurons, β-actin is enriched in immature growing axons relative to the cell body and γ-actin is uniformly distributed throughout the cell (19, 63). In the gastric epithelium, β-actin localizes preferentially at the apical membrane of parietal cells, whereas γ-actin is found at both membranes and predominates at the basolateral membrane (64, 65). In ciliated and goblet cells of human airways and in SPOC1 cells, β- and γ-actin appeared to colocalize strongly to the apical membrane (Figs. 2 and 3). Although it is tempting to speculate that the differential distribution of β- and γ-actin in many cells may mean that they subserve different functions, in fact such discrimination between isoforms has yet to be demonstrated. At least in experimental situations, the two isoforms appear to function indiscriminately in homogenous or mixed populations in actin filaments (27).
It is widely accepted that the basic engine for cell motility is the actin cytoskeleton, an engine composed of closely coordinated actin filament polymerization and degradation, cell adhesion, and membrane remodeling. Intense research efforts during the past two decades have revealed the formation of protrusive structures involved in motility to be due to actin polymerization after the activation of the Wiscott-Aldrich syndrome (WASp/SCAR) family by Rho GTPases (Cdc42, Rac), phosphatidylinositol 4,5-bisphosphate, or several of the Src homology domain 3 proteins such as Grb2, followed by filament branching, nucleation, and extension under the influence of WASp/SCAR and actin-related protein 2/3 complex (45, 46). The polymerizing actin filaments, anchored within the leading edge, provide a motive force to extend the plasma membrane outward from the cell (see Ref. 36). Interestingly, this same actin polymerization machinery has been implicated in intracellular organelle transport (26, 35a), endocytosis (4), and various aspects of exocytosis (see below).
The role of actin filaments in exocytosis is controversial, which is to say that actin potentially has multiple functions in secretion and/or that the precise mechanisms may be cell-type dependent. Classically, the actin cortex has been viewed as a barrier that impedes the movement of secretory granules to the plasma membrane (7, 30a, 57a, 62); however, in other cells such as PC12 and insulin-secreting cells, dynamic actin remodeling may be required for the exocytotic process (17; 30b; 35b). Recent biophysical studies within the near membrane environment of secretory cells suggest that actin polymerization is required for exocytosis as though the secretory granules were being guided actively to plasma membrane docking sites (40). A key distinguishing feature between these barrier and transport roles, experimentally, is the effect of cytochalasin D and/or latrunculin on secretion. In studies favoring a positive role for actin filaments in secretion, disruption was inhibitory (44), whereas disruption was stimulatory in studies suggesting a negative barrier role (37). In addition to barrier and transport roles, actin filaments have also been shown in Xenopus oocytes and pancreatic acinar cells to polymerize around secretory granules after exocytotic fusion. These new filaments function to provide the motive force for expulsion of granule contents and/or to maintain the "Ω-figure" as a prelude to endocytotic membrane retrieval (39, 56). In both cases, latrunculin inhibited actin filament coat formation around the granules and promoted fusion of adjacent granules. Latrunculin, however, either had no effect on exocytosis or was stimulatory in these cells. Hence, actin filaments may function variously to regulate exocytosis negatively in resting cells, to guide granules to docking sites after cell stimulation, and/or to stabilize Ω-figures after exocytosis.
In goblet cells and SPOC1 cells, neither β- nor γ-actin could be identified around the mucin granules near the apical membrane or anywhere within the mucin granule region of the apical pole; rather, actin was positioned prominently as a thick sheet between the granules and the apical membrane (Figs. 2 and 3). After stimulation, both isoforms disappeared from the SPOC1 cell apical membrane domain (Fig. 3B, right). Latrunculin A increased basal secretion (Fig. 6), suggesting that disruption of actin filaments alone is sufficient to induce secretion. Furthermore, agonist-stimulated SPOC1 cell mucin secretion was enhanced, indicating that even in stimulated cells, actin filaments act to regulate granule release negatively (Fig. 6). Additional evidence of a barrier role for actin in SPOC1 cells includes the inhibition of mucin secretion in activated cells by filament stabilization using jasplakinolide (Fig. 7) and by overexpression of actin isoforms (Fig. 8). In addition, the two proteins hypothesized to effect disruption of the actin barrier in secretory cells, MARCKS and scinderin (58, 61), are not only expressed in SPOC1 cells but also appear to act in accordance with the barrier hypothesis after cell activation. MARCKS is phosphorylated, indicating a translocation to the cytosol, and scinderin appears to sever actin filaments (Figs. 4 and 5, respectively). Hence, several lines of independent evidence strongly support a barrier role for actin filaments in regulated mucin secretion from SPOC1 cells. Two aspects of the data suggest that it is the predominant function. First, the apparent lack of actin within the mucin granule region makes an actin-based granule transport mechanism difficult to envision. Second, the stimulation of secretion in resting cells by latrunculin A suggests that actin filament disruption alone may be sufficient to trigger the exocytotic process. Consequently, if actin-based mucin granule transport does occur in goblet cells, it may not be a prerequisite for secretion as it is in other cells (17, 41).
The role of MARCKS in regulated exocytosis in goblet cells is presently controversial. Recent data showing inhibition of mucin secretion by a myristoylated NH2-terminal MARCKS peptide led to the novel hypothesis that the protein plays a role in a transport of granules by actin filament from the cytosol to the apical membrane (32, 55). Our data regarding MARCKS phosphorylation suggest that translocation from the membrane to cytosolic fraction upon agonist stimulation (Fig. 4) and stimulation of mucin secretion by latrunculin A (Fig. 6) are more consistent with the barrier hypothesis (60). The current data do not rule out a granule transport role for MARCKS; however, our data do suggest that if MARCKS plays a role in mucin granule transport, it is likely to be subservient to the barrier function.
Interesting possibilities potentially reconciling the apparent contradictory roles of actin filaments in secretion are that their barrier and transport roles are interwoven and that a factor determining which role dominates in a particular cell type is granule size. Secretory granules generally range in size from ∼50 nm to >1 μm in diameter, and mucin granules at 0.8–1.5 μm (13, 31) are among the largest known. Larger-diameter granules may have an increased likelihood of interaction with plasma membrane docking sites because of the greater surface area and smaller angle of curvature of the limiting membrane. For the smallest granules, positioning by actin filaments to such plasma membrane sites may be necessary to ensure a dynamic secretory response. From a regulatory point of view, a barrier to secretion formed by actin filaments is a very attractive notion: secretory granules are retained in the cells at low energetic expense by the actin barrier as they mature and the cell awaits activation, and they gain rapid access to exocytotic docking sites while the actin filaments are remodeled dynamically upon receipt of an appropriate signal.
The present data do not allow us to draw conclusions regarding the possibility of a postexocytotic role for actin filaments in supporting Ω-figures in mucin-secreting cells as revealed recently in other cells (5, 39). It is interesting to note, however, that because mucin granules in airway goblet cells are exocytosed completely in <100 ms (13), such a role may be unnecessary.
To conclude, β- and γ-actin participate in the formation in airway goblet cells of an apical actin cap that acts as a physical barrier to mucin secretion under resting conditions. Upon stimulation, the actin filaments rearrange under the actions of MARCKS and scinderin to allow granule access to the plasma membrane. However, it is likely an oversimplification of what really happens in regulated mucin secretion to think that actin filaments simply require disassembly to provide access to docking sites for mucin granule exocytosis. Further studies are required to investigate whether actin filament disassembly in the apical region is directed or random, whether β- and γ-actin form independent isoform-specific filaments that depend on different regulatory systems, whether local actin polymerization operates secondary to disruption to guide granules to exocytotic sites, and the specific roles of PKC and MARCKS as well as Ca2+ and scinderin in regulating and/or effecting actin filament remodeling.
K. De Pestel acknowledges support for the development of the scinderin-specific antibody by Grant FWO-G.0007.03 (to C. Ampe, Department of Biochemistry) and Grant GOA 12051401 of the Concerted Research Actions of the Flemish Community (to J. Vandekerckhove and C. Ampe). Primary support for these studies was derived from grants to C. Ehre from La Foundation Vaincre la Mucoviscidose, Paris, France, from National Heart, Lung, and Blood Institute Grant HL-63756 (to C. W. Davis), and from the North American Cystic Fibrosis Foundation.
We are grateful to Mireille Ladislas, Novartis Horsham Research Centre, for expert technical assistance in the cloning and analysis of human scinderin and to Drs. Michael Chua and Silvia Kreda for expert assistance in microscopy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society


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Hidden Talents - Local amateurs take on the piano classics, presented by LocalEyes
Gualala Arts Center
Gualala, CA
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Hidden Talents - Local amateurs take on the piano classics, presented by LocalEyes
Four local pianists - Judy Biehler, Bette Covington, Paula Osborne and Roland Stoughton - will give 15 minute solo recitals, presenting works by Bach, Brahms, Schumann, Chopin, Debussy, Grieg, Janáček and Prokofiev. Refreshments and conversations with the musicians follow the performance. The program will provide diversity of music and of musical approach.
The word 'amateur' in the title of this event does not apply equally or precisely to each of the four performers. For example, Ms. Covington taught piano privately for 30 years, and she and Ms. Biehler and Ms. Osborne have fairly extensive performance experience. Both Ms. Covington and Ms. Osborne were long time students of Roy Bogas, who local concert goers will know from his frequent Chamber Music Series appearances at the Gualala Arts Center. However, each of the four would eagerly admit the gap between them and concert artists who have pursued performance for a living and made it the highest life priority.
Most members of the audience will remember some personal experience with performing classical music as a child, and will appreciate how difficult this is; the event should be even more enjoyable for this empathy. Be glad that they are up there and you are in the audience! Mr. Stoughton, who began serious piano study only after retiring to The Sea Ranch in 2010, recalls one of his twin sons remarking after their first childhood recital "I felt like my teeth were going to fall out."
Classical piano is often a lonely endeavor. Amateur orchestras do not typically include piano, and chamber music (classical music for small ensembles) almost always requires a very high level of skill to undertake it. Unlike most other instruments, piano music contains multiple voices at once which have to be learned, so it is particularly difficult to break into chamber music as a pianist. The violinist and cellist are always turning to you and saying "is that ALL you've learned since last week?!" Hence the predominance in classical piano of solo music, solo performance and solo practice! This event is a great opportunity for the performers to share these efforts, and some wonderful music, with their community.
Performer Biographies
Judy Biehler began piano study after a childhood encounter with a broken glass dish caused a serious hand injury. The doctor suggested playing the piano would be good hand therapy. She began her study with a fine teacher in Chico, Kay Thompson, and studied with her for over 10 years. She also played the clarinet during this time, participating in symphonic and marching bands. At CSU Chico, she continued her piano study with C. Robert Laxson and studied the organ with Kay Thompson. Under Kay's tutelage, she began teaching piano. After college, she studied for 12 years with Frank Wiens, Professor of Piano at Stockton's University of the Pacific and performed in numerous chamber ensembles and had a long duo piano partnership. She has most recently studied for two years with Professor John Milbauer, now teaching at the University of Arizona at Tuscon and during the summers, at Chataqua, NY. She has performed in master classes including ones by Karl Ulrich Schnabel. She enjoys being an artist as well, where "performance" is quietly on paper and canvas.
Bette Covington grew up in Claremont, California, in a family where each of the five children was required to study a musical instrument. She began her piano studies at the age of 5 and continued until she graduated from high school as a promise to her father. During her four years at Stanford University she sang in the choir and then focused, with her husband, Marty, on raising their two sons, teaching 5th grade in Orinda, and maintaining a piano studio in Moraga where she taught privately for thirty years. During those thirty years she resumed her piano studies, as a long time student of Roy Bogas and also occasionally with Steven Gordon in New York. Bette was an active and performing member of the prestigious Berkeley Piano Club, the California Music Teachers Association, and the Etude Club of Berkeley. After moving full time to her Sea Ranch home in 2003, Bette closed her piano studio but continues to teach and to perform on occasion. Her interests have moved into the realm of fiber arts and she enjoys designing and making quilts, both for entries in local shows and for comfort quilts to the community.
As a child in Indianapolis, Paula Osborne was drawn to the piano and played by ear the pieces her older sister was learning. When Paula was 5 years old her elders decided that she should learn to read music instead, and her education began. She continued to study piano through high school and college, and after a 13-year hiatus from the piano while running her travel agency in Estes Park CO, she returned to the keyboard in earnest. Paula performed several solo recitals there before moving to the Bay Area, where she was fortunate to study with Roy Bogas in Berkeley for over a dozen years. Since moving to The Sea Ranch she has enjoyed playing locally for her own pleasure and that of her friends.
Roland Stoughton received the usual ineffectual intermittent suburban piano instruction as a child, and then was fortunate to have one good year at Smith College Conservatory while a student at nearby Amherst College. Although career and family subsequently nixed piano for 35 years, that year planted the idea of returning to it someday, which Stoughton was finally able to do when he retired to The Sea Ranch in 2010. Stoughton currently studies with Nancy Ohlbach of San Francisco. Stoughton also sings in the Mendonoma Madrigal group. Stoughton has a PhD in Physics, many publications in the fields of astrophysics, geophysics, defense systems analysis, and biotechnology, is author of 30 patents in biotechnology and the founder of two successful biotechnology companies.
Tickets are $10 in advance, $5 more on the day of the performance. Youth 7 to 17 are admitted free. To purchase in person, visit the Gualala Arts Center or Dolphin Gallery in Gualala.
Gualala Arts Center
46501 Gualala Road
Gualala, CA 95445
United States
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subset: commoncrawl


text: Glad it worked out and you got your Wii back. I don't believe you used the waninkoko updater-at least according to your posts. Of course, I'm sure you didn;t spill all your secrets in the posts. Anyway, happy it worked out for you. Go back to enjoying your Wii and don't accept any updates until you check this site.


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The Korner Klub May Close – NO!!!
I live at Four Corners and I eat at the Korner Klub probably once a week! I would be so bummed if they close!!! ~Erin
Four Corners restaurant might have to close because of concerns about its septic system.
The Korner Klub was denied a restaurant license because it doesn't have a permit for its public wastewater system, the Gallatin City-County Health Board decided Friday.


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Andrew: I let Johnson down
Rob Andrew has admitted he failed Martin Johnson during his tenure as England manager.
Last Updated: 25/11/11 8:52am
Director of elite rugby Rob Andrew has admitted he failed Martin Johnson during his tenure as England manager.
The pressure on Andrew has intensified since the publication of excerpts from three leaked World Cup reviews.
The findings paint the picture of a dysfunctional England squad and a coaching team out of its depth.
Johnson resigned as the team manager last week and attack coach Brian Smith, criticised heavily by some players in the leaked report, has also stood down.
Andrew described the Rugby Football Union as being at "rock bottom".
"I am absolutely shattered by what is going on both on and off the field," said Andrew.
"The RFU has to sort itself out. This is rock bottom, the lowest of the low, and it can't be allowed to continue.
"The reality of my job is that I am not the England team manager. I am the elite rugby director and I run a big department. But I absolutely accept responsibility for what is going on here.
"The World Cup was not what it should have been. The players have let themselves down, the coaches have maybe not done as good a job as they should, I've not done as good a job as I should have done.
"I should have supported Martin more and we should have done better."
The revelations have piled pressure on Andrew to resign but the former England fly-half has refused to step down.
"Of course I've considered my position. There's been soul-searching the whole time, it's been a pretty tough 12 months for everybody here.
"Does it mean I'm going to resign? No, I'm not. Does it mean it's all broken? No, it doesn't."
There is no financial incentive for Andrew to step down. It is understood he would receive the equivalent of six months' salary if he resigned but if the RFU wanted to remove him it could cost them at least three times that.


text: I am looking for pictures of a sub box that does not disrupt the kick panel/foot rest under the help. My dad is a huge fan of the diagonal foot rest and in order for me to put the sub in the boat, he won't let me take away that space. I am putting a 12" JL W6v2 that requires a 1.25 sqft sealed box. I have searched the forums for other pictures but really only found set ups using a faux wall and eliminating the foot rest all together. Has anyone seen this done or have pictures?


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A team of academic researchers have developed a pair of tools which combine to improve web browser speeds by over 30 percent, according to a research paper released this week.
Ravi Netravali, Ameesh Goyal, and Hari Balakrishnan from the Computer Science and Artificial Intelligence Laboratory (CSAIL) at MIT and James Mickens of Harvard University will present the findings published in "Polaris: Faster Page Loads Using Fine-grained Dependency Tracking" (PDF) at the USENIX Symposium on Networked System Design and Implimentation next week.
Browsers traditionally gather the elements of a page in the order determined by a dependency graph, but hidden dependencies force them to make conservative assumptions about that order, making inefficient use of network and CPU resources. The researchers addressed this inefficiency by developing "Scout" to capture edge dependencies by tracking fine-grained data flows, and dynamic client-side scheduler "Polaris" to load objects in an order determined by fine-grained dependency graphs.
Read more: As Part of Probe, NY State Attorney General Invites Public to Test Internet Speed
"The objects in a web page can interact in complex and subtle ways; however, those subtle interactions are only partially captured by lexical relationships between HTML tags," the researchers said in the report. "Unfortunately, prior load schedulers have used those lexical relationships to extract dependency graphs. The resulting graphs are underspecified and omit important edges."
The researchers explain the conservatism browsers are forced into by partially revealed, coarse-grained dependency graphs with the example of browser evaluation of first.js tag, which may or may not modify downstream objects.
The researchers also told Mashable that although it has yet to be tested with various browsers, Polaris is in principle browser-agnostic, and also that there is no increased risk of loading malicious code or malware associated with it.
Most of the effort into improving the speed of websites seems to focus on the server side, where there are a number of measures site operators and web hosts can take. Load times for many pages can also be improved with adaptive database engines.
Google discontinued its PageSpeed service last year, leaving the market to CDNs like Cloudflare.


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The bodacious victory of Ted Cruz in the Texas Republican primary has somehow fired up Texas Gov. Rick Perry, whose own right-hand man lost to Mr. Cruz on Tuesday by 14 fat percentage points. But the ever-canny Mr. Perry — who was leading the presidential polls only a year ago — has ridden the Cruz victory like a bronco, tamed his own disappointment and framed the Lone Star State in heroic terms.
"Ted is a force to be reckoned with, an excellent candidate and a great conservative communicator. I call on all conservative Texans to rally behind Ted in November so we can remake the U.S. Senate in the image of Texas for the good of all Americans," Mr. Perry exclaims. "Lt. Gov. David Dewhurst fought a valiant fight. I'm proud to call David my friend. He and I will stand shoulder to shoulder once again in the spring, fighting for Texas, the most conservative state in America. Texas is as rock-hard conservative as ever."
"Flipping the bird to Rahm: Chicago Chick-Fil-A mobbed," reads the headline from "Instapundit" Glenn Reynolds of PJ Media, who was among many who chronicled Mike Huckabee's "Chick-fil-A Appreciation Day" on Wednesday. Yes, thousands turned out to tuck into crispy chicken and waffle fries from the embattled restaurant chain in dozens of locations; there were traffic jams in South Carolina and long lines in the Windy City, where Mayor Rahm Emanuel himself was among politicians vexed by the eatery's support of traditional marriage.
Like their tea party brethren, the Chic-Fil-ites proved a cheerful, well-behaved bunch when it came to staging public demonstrations. And conscientious, too.
"One woman and her friend took up a collection at their office to buy bags of the chicken sandwiches to deliver to an area homeless shelter," reports Anne Sorock, who manned one lengthy line in midtown Chicago, and is a contributor to the blog LegalInsurrection.com.
"Let God plan parenthood."
(Bumper sticker spotted in the District.)
That would be the Twitter Political Index — or Twindex — a gauge of fickle public sentiment toward President Obama and Mitt Romney, at least according to the number of daily tweets sent on each candidate. Launched Wednesday, the Twindex is a project of Twitter, the search engine Topsy and a partnership between bipartisan pollsters Mark Mellman and Jon McHenry. See it here: election.twitter.com.
Religious freedom: It was a big deal for the Founding Fathers, and bolsters the very soul of the nation. And while the cause gets short shrift in the media, it warrants some steady attention on Capitol Hill, thanks to a series of ongoing gatherings of lawmakers, experts, eye witnesses and activists eager to register on the public radar. Such was the case at a briefing Wednesday titled "America's Response to Religious Persecution in Allied Nations" at the U.S. Capitol Visitor Center, staged just a hallowed hallway away from hundreds of awestruck tourists seeking a patriotic fix.
In the hush of the briefing room, a spate of speakers that included Rep. Gus M. Bilirakis, Florida Republican; Rep. Danny K. Davis, Illinois Democrat; Kathryn Cameron Porter, founder of the Leadership Council for Human Rights and the Rev. In Jin Moon, a human rights activist and president of the Unification Church USA, weighed in on the big question: Is the U.S. paying attention to persecution abroad? Well, yes, and no. The issue is still a work in progress.
"There is no real definition of religious freedom around the world," said Tina Ramirez, director of international relations of the Becket Fund for Religious Liberty, who recommends that the State Department effectively, fearlessly articulate some exacting parameters, and for a global audience.
"Many of America's democratic allies profess to uphold human rights and religious freedom, but in reality they do not. The U.S. should confront their violations," said Aaron Rhodes, co-founder of the Freedom Rights Project, a research group. He too hopes the U.S. government steps up to the plate and demands "international human rights standards." But it is diplomacy that demands finesse.
"We have to start by listening, and work together for a change," Mr. Rhodes said, recommending the U.S. concentrate on the aforementioned standards, rather than assuming the position of a "role model" in an oft contentious, emotionally driven dialogue.
Now emerging after Mitt Romney's emphatic trip to Israel: That would be the Jewish Americans for Romney coalition, which counts among its honorary chairmen Rep. Eric Cantor of Virginia and former Sens. Norm Coleman and Rudy Boschwitz of Minnesota, plus a vast advisory board that includes Tevi Troy, a senior adviser to former President George W. Bush.
"I urge all American Jews — Democrat, Republican and independent alike — to give a serious look at Mitt Romney's candidacy. Throughout his life, Gov. Romney has been an unwavering supporter of the state of Israel. As he stated during his most recent trip to Jerusalem, 'By history and by conviction, our two countries are bound together,'" Mr. Cantor says.
"Mitt Romney intends to send a signal to the world, and especially to Israel's adversaries, that the United States is not a fair-weather friend of Israel, but a partner in an abiding relationship based upon a common commitment to our most fundamental values," says Mr. Coleman.
• 48 percent of Americans favor gay marriage.
• 24 percent of Republicans, 17 percent of conservative Republicans, 21 percent of tea party supporters, 65 percent of Democrats and 80 percent of liberal Democrats agree.
• 33 percent of Protestants, 19 percent of evangelicals and 53 percent of Catholics also favor gay marriage.
• 44 percent of Americans overall oppose gay marriage.
• 70 percent of Republicans, 76 percent of conservative Republicans, 71 percent of tea party supporters, 29 percent of Democrats and 22 percent of liberal Democrats agree.
• 58 percent of Protestants, 76 percent of evangelicals and 37 percent of Catholics also oppose gay marriage.
Source: The Pew Forum on Religion & Public Life survey of 2,973 U.S. adults conducted June 28 to July 9 and released Tuesday.
• Soliloquies, jaunty one-liners to [email protected]
Copyright © 2017 The Washington Times, LLC.


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A note to my fellow forum members on answering questions.
If you don't have a clear view on the situation surrounding a question please consider it your responsability to get that first before answering questions in a way that will degrade security without strongly suggesting any secure alternative.
I would even like to go further and deny the answer unless the user *really understands* the consequences.
After all this is a security forum and I think our goal should be to *help* ppl who enhance or maintain security and *educate* those that want to open up holes w/o explaining or bypass std security measures w/o understanding the consequences.
I think this is a good idea too. Even if you're on a private network, it's still not the greatest idea to bypass security. It makes it easier if you decide to turn your private network wireless or some weenie tries to hack you from the inside.
|All times are GMT -5. The time now is 05:10 PM.


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Marinara di Venezia
For countless years in countless Italian towns, countless Italian cooks have prepared spaghetti sauce just like this — full of chunky red tomatoes and seasoned with oregano, marjoram and parsley. Venice, one of our favorite Italian cities, inspired our recipe. But we encourage you to add ingredients and put your own twist on it. Then hand down the recipe to (countless) generations of your own.
Ingredients Tomatoes*, tomato puree*, onions*, extra virgin olive oil*, sea salt, parsley*, locust bean gum*, basil flavor, oregano*, black pepper*, garlic flavor*, marjoram*.
* = Certified Organic


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- J. A. White,
- L. C. Munn and
- S. E. Williams
Three Aridisols and an Entisol in the Red Desert of southwest Wyoming were surveyed for vesicular-arbuscular mycorrhizal (VAM) fungal biomass. The survey was conducted to determine attenuation of VAM fungal biomass in profiles of these arid soils, to examine possible edaphic influence on VAM fungal distributions and to evaluate the potential of these soils in providing VAM fungal biomass for use in reclamation. Fungal biomass levels—as indicated by fungal spore densities and bioassay percent infections—was apparently related to morphological and chemical properties that differ between individual soils and their associated vegetation. While fungal spore densities generally attenuated with depth in the Aridisols, the Torrifluvent retained high densities of spores (up to 81 spores g−1 soil) to a depth of 140 cm. The VAM fungal biomass attenuation in relatively deep horizons of these arid soils may still allow adequate formation of propagules for use in reclamation of disturbed lands.Please view the pdf by using the Full Text (PDF) link under 'View' to the left.
Copyright © .


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Originally posted by Humboldt9er:GTFO of here. montana built steve youngs team how? by being there first? by drafting all of those players? by coaching them up? by playing defense? its a team game, montana didnt build s**t. he played on the team and he played awesome but he didnt build s**t. that was one of the dumbest statements ive ever read on here. thank you for that
Originally posted by 49ersalldaway126:
more linke since mooch left
since mooch left? na. more like since the niners thought steve young was a better option than montana. i loved watching the cheifs beat the niners that day. its been down hill since. montana buildt youngs ring,
There are 111 users in the forums
harbaugh vs raye
Sep 22, 2011 at 12:34 AM
- hondakillerzx
- Veteran
- Posts: 16,994
Sep 25, 2011 at 2:21 PM
- GolittaCamper
- Veteran
- Posts: 6,686
The offense is growing, but certain players lake of skill and discipline is holding back the development! I did see some things I liked today, just some poor execution by players that seem to have forgotten how to win!


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Here's an advance look at my review of African American Repertory Theater's The Nacirema Society...:
Pearl Cleage's latest comedy is set in the 1960s. It feels as if it was written even before that — as light and fluffy as the trivial pieces little theaters used to mount in the 1950s.
African American Repertory Theater's production of — note the long-winded title — The Nacirema Society Requests the Honor of Your Presence at a Celebration of Their First One Hundred Years doesn't help matters. Director Ptosha Storey allows her actors, many of them extremely talented, to run wild with exaggerated silliness and portentous line readings. Once again, we're back in the bad old days when staginess substituted for reality onstage.
The Nacirema Society might remind you of The Last Night of Ballyhoo, only set in a black Southern community rather than a Jewish one. Shut out of white high society back in the days of Jim Crow, wealthy women of color in Montgomery, Ala., have created their own debutante parties. The reigning queen bee, Grace Dunbar (Cheryl Tyre), is eager to show off her granddaughter, Gracie (Perri' Camper). Grace's daughter-in-law, Marie (Catherine Whiteman), is more restrained and sensible about the whole thing.
Grace and old friend Catherine Green (Tippi Hunter) are conspiring to pair up Gracie with Catherine's grandson, Bobby (Christopher Dontrell Piper). Gracie and Bobby, however, are more like brother and sister. Catherine, despite Grace's initial reservations, has granted an interview to a New York reporter (Nadine Marissa) who had previously published a less than flattering article about the Nacirema Society. The daughter of the Dunbar family's late housekeeper, Alpha Campbell Jackson (Regina Washington), also intrudes into the festivities with unwelcome news, although her brilliant daughter, Lillie (Whitney Coulter), opposes Alpha's nefarious plans.
It's Tyre and Hunter who most frequently get out of hand stylistically. The actors playing the younger characters are far more committed to verisimilitude. Tyre assumes poses and utters line with the panache of stars of an older generation: Split the difference between Kathleen Turner and Tallulah Bankhead. Hunter approaches physical comedy like a minor character from an old-fashioned sitcom, loose-limbed and stumbling even when she's not supposed to have drunk too much sherry.
Cleage does pay lip service to the bigger issues of the era. Her younger characters are working in the Civil Rights movement. Basically, though, The Nacirema Society is feather-light and none too plausible.
Plan your life
Through Feb. 24 at the DeSoto Corner Theater, 211 E. Pleasant Run, DeSoto. Runs 160 mins. $20 to $25. 972-572-0998. aareptheater.com.


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Thin-film solar panel maker MiaSole has reportedly raised $125 million in sixth round of equity financing.
The round values the company at less than half it was in 2008, dropping from a $1.2 billion pre-money valuation in 2008 to a $550 million valuation today, according to a source cited by VentureWire.
The company has been said to be preparing for an IPO this year (and was last reported to be looking to raise $100 million, so if this report is correct, it beat that goal). It was also recently sued in a patent infringement suit. MiaSole's investors include Kleiner Perkins Caufield & Byers, VantagePoint, and Firelake Capital Management.
Silicon Valley solar panel companies had a buoyant period of investor interest around 2008, when companies like Solyndra, MiaSole and NanoSolar were able to raise hundreds of millions of dollars. Times have since gotten tougher. Solyndra shut down its first factory ahead of schedule in order to cut costs. Some solar companies (not all venture-backed) like Evergreen, Tessera and SpectraWatt have been forced to layoff workers and shut down projects and plants, and others, like Applied Materials and Suntech's thin-film lines, were closed entirely.
MiaSole's ability to raise this much in this environment is certainly a good sign, and even the lowered valuation is still a good one. The company recently reported reaching a bar-setting CIGS efficiency rate of 15.7 percent. And despite some struggles in solar, solar companies in thin-film and the CIGS technology that MiaSole makes have seen a lot of good news recently, with Stion, SoloPower and AQT Solar all announcing plans to build solar factories.
Makers of CIGS panels, named for their ingredients of copper, indium, gallium, and selenide, are expected to cut costs drastically and improve efficiency in the next few years, bringing it closer to the efficiency rates of the dominant (and currently Chinese-dominated) crystalline silicon technology. Its customers include SolarCity, Wal-Mart, Chevron, and Juwi.


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|low graphics version | feedback | help
|You are in: Business
Wednesday, 15 November, 2000, 20:48 GMT
E-tail growth hits out-of-town stores
The growth of internet shopping is hitting the value of out-of-town retail centres, according to the UK's largest listed property firm.
The internet is, by enabling easy comparisons between different outlets, deterring retailers from increasing prices, and so squeezing margins, the firm's chief executive Ian Henderson said.
"What you are finding is that the branded names are not so badly affected, because people will still be prepared to pay for them," Mr Henderson told BBC News Online.
"Discount stores are also not so badly affected. It is the middle area of retail which is being the worst hit."
Company revamp
The firm credited a strategy imposed in May of improving revenue, and selling assets which could not be further developed, for preventing a greater decline in the value of retail assets.
"The relatively small adverse movement in value... shows the success we have had in implementing our rationalisation policy," the firm said in its results statement for the six months to the end of September.
Shopping sites outside prime areas were being worst hit as retailers "rationalise their space requirements", the statement said.
Shopping centres 'will survive'
But Mr Henderson did not foresee the slide heralding the end of the out-of-town shopping centre.
"But what people will also want to do is be able to pop round the corner, to pay for convenience."
The firm's investment in city centre developments, typically containing retail and office units, aims to exploit this trend, he said.
Huge portfolio
The value of the company's overall property holdings rose 7% year-on-year to Ł7.73bn, with growth in the London portfolios, including New Scotland Yard and much of Oxford Street, more than offsetting the squeeze on shopping centre sites.
But while rental income rose slightly to Ł243m, costs including Ł97.2m development spending saw pre-tax profits decline 2% to Ł148.5m for the six months.
The firm will, within the next few months, discover whether it has, with partners, won contracts to run London Underground and BBC property portfolios.
Shares in Land Securities closed 11.5 pence higher at 782.5p in London on Wednesday, valuing the firm at less than two-thirds of its property holdings.
See also:
07 Nov 00 | Business
Christmas cracker for web retail
24 Oct 00 | Business
Housing market 'stable'
03 Nov 00 | Business
House prices slip
16 Oct 00 | Business
Traditional retailers beat dot.coms
Internet links:
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Bulkypix and Anima Game Studio are teaming up for an iPhone, iPad and iPod touch launch of Ark of Sinners Advance on April 18th. This is sure to please fans of the Anima Beyond Fantasy franchise, as well as fans of action/platformers in general and of women who don't understand the proper use of shirts.
For those outside the know, Anima Beyond Fantasy is a popular franchise of role-playing books, miniature wargames and card games that have become a success at the critic and commercial levels. Ark of Sinners Advance is an iOS action/platformer based on the franchise. In it, you'll play as Celia, a powerful warrior with a past so mysterious even she doesn't know what's going on. She was adopted as a child by a secret society of assassins who trained her to become "…a perfect fighter that far exceeds human limits," but who forget to show her how to properly dress for an afternoon of hacking and slashing.
After refusing to kill a renegade agent, Celia finds herself on the run with Nemo, her only remaining friend, and must journey to the depths of the ancient city, Ark, to discover its sinister secrets.
Ark of Sinners will feature:
- 21 interconnected stages that will offer more than 10 hours of gameplay.
- Countless combat movements.
- 18 Stones of Knowledge hidden along the city to unlock a secret ending.
- Exciting adversaries. 14 different enemies and 7 bosses.
- Epic music. Enjoy a wonderful orchestral soundtrack.
- A deep and intricate storyline: Reveal the secrets of Ark trough the tales of Carlos Garcia (Anima Beyond Fantasy series writer)
- Artistic Direction and animated cutscenes from the well-known artist Wen Yu Li.
- Retina compatibility
Ark of Sinners will be available for iPhone, iPad and iPod touch on April 18th for $2.99 | 2.79€. For more information, visit bulkypix.com.


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Jan - thank you, I'm so happy that you enjoy this too. I liked how this turned out - the shiny and 3D effects surprised me, but then we all like nice digital surprises!PJ
Mandy - thank you, I'm so delighted you enjoyed it. It is exciting to try out new digital tools. Yes, I do knit too - every week. Quite a few Norwegians do knit, a tradition over here. PJ
The central image with such shiny swirls around it. So beautiful, PJ! thanks so much! Love the 3D effect on this one!!
PJ, I love the way the centre of this has so much texture - and I bet you don't get lost when you're wearing this hat... the traditional knitted woollens finally seem to be coming back into fashion, for which I'm delighted... do you knit as well as embroider??? Thanks for another lovely puzzle :~)
Thank you Kathie - I'm so glad you enjoyed it. The snowflake pattern is part of a knitted wollen hat (old traditional pattern). I'm trying out some new digital tools....PJ
I love this orange and yellow fire wheel! Excellent snowflake design in the middle too! Thanks PJ.


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Berlin, Oct 7 (IANS) German industrial output fell in August, official data showed on Wednesday, one day after reports that the country's industrial new orders slumped for two months in a row.
Compared with the previous month, German industrial output decreased by 1.2 percent in August, following a monthly increase of 1.2 percent in July, Xinhua quoted German federal statistical office Destatis as saying.
Economists had expected the output to inch upward by 0.2 percent in August.
According to Destatis, the drop was led by production of capital goods such as machine and equipment which fell by 2.1 percent.
"The unfavourable configuration of vacation days has significantly contributed to the decline in production," the German Economy Ministry said in a statement.
In a less volatile three-month comparison, the average industrial production in the past three months increased slightly by 0.2 percent.
The output data came one day after reports that new orders received by German factories fell for second consecutive month in August by 1.8 percent due to weak demand from home market and countries outside the euro zone.
The ministry, however, predicted that German industrial activity remained "moderately positive" thanks to robust business confidence and export development.


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Millennials may spend their lives connected via Facebook, Twitter, and SMS, but that doesn't mean they automatically know how to produce multimedia journalism.
In fact, most student media organizations still work out of separate offices, with the student paper, radio station, and TV staffs rarely working together.
That's part of the reason why Texas Christian University's Schieffer School of Journalism opened a $5.6 million "Convergence Center" last year, writes Aaron Chimbel at the Online Journalism Review. Now the newspaper, magazine, and TV station are in the same building, bouncing ideas off each other.
"Because News Now [the station] and Skiff [the newspaper] staffers were working in the same newsroom, we were much more aware of what the other one was doing than we were before," Julieta Chiquillo, the Skiff's managing editor in fall 2009 and editor-in-chief the following semester, told Chimbel. "Even then, we had to establish a system to better communicate." And there's a feeling of "competition" because of course the paper still wants to scoop the station and vice versa.
But they're learning. It helps that the school's done away with separate degrees in broadcast or print journalism and now just offers one program. A reporter last year did a Web video and text story which eventually became also a print piece in the paper and a segment on News Now.
The danger, of course, is the same danger at any organization that's using pools–you have one reporter now expected to notice everything, rather than many reporters all pursuing different angles of the same story.
But let those who've graduated figure that out. For now, it sounds like the students are learning actual 21st-century skills from a university. Shocking.


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BAL HARBOUR, Fla., Jan. 1 (UPI) -- Louis Wolfson, a college dropout who became a multi-millionaire businessman and racehorse owner before going to prison, has died at 95 in Florida.
Wolfson suffered from colon cancer and Alzheimer's disease, the Miami Herald reported. He died Sunday on the 30th anniversary of his marriage to Patrice Jacobs, daughter of a prominent racing trainer.
A millionaire by the time he was 28, Wolfson was first involved in construction and shipbuilding. In 1960, he founded Harbor View Farm in Florida's Marion County and bred generations of thoroughbred horses, including Affirmed, winner of the 1978 Triple Crown.
In business, Wolfson was chairman of the company that built the Glen Canyon Dam and also headed the company that produced Mel Brooks and Woody Allen movies.
In the 1960s, Wolfson was jailed for almost a year for selling unregistered stock. He always believed he had been railroaded.
"I didn't use any fictitious names in selling the stock. No Swiss banks,"' he said. "When I made the sale, I reported it to the SEC. I'd have had to be an idiot to do anything willfully wrong."
After his release, he became a crusader for prison reform.


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Los Angeles Clippers
By Ben Zani
RotoWire Writer
In a single step, the 2007-08 Clippers season ended before it even began. That step, of course, was the one taken by All-Star forward Elton Brand that tore his left Achilles tendon, sidelining him for the foreseeable future and ending any hopes that the Clippers have of being competitive this season. Brand was the backbone of this Clipper team, and without him, it will probably be another lottery-bound year for Los Angeles' other team.
Even with Brand, last year's Clippers were among the league's biggest disappointments, finishing out of the playoffs and seven games off of the 47-35 club that came within a game of the Western Conference Finals a year earlier. Last year's Clippers were poor at best, ranking 23rd in the league in scoring and 19th in field goal percentage. They finished at or near the middle of the pack in every other statistical category save for blocked shots, where the Clippers' 5.8 per game tied for first league-wide with Detroit. Their leading shot blocker last year however was Brand, who will now miss most of the season. Brand in fact led last year's Clippers in minutes, scoring and rebounding, and was second to little-used Aaron Williams in field-goal percentage. The main priority of this year's Clipper team will be to somehow maintain without their unquestioned leader.
Unfortunately, there are not many candidates to fill Brand's sizable shoes. Sixth man Corey Maggette was second on the team in scoring with 16.9 points per game, and could pick up a bit more of the scoring load. Center Chris Kaman was second to Brand with 7.8 rebounds per game, but much of his success was due in part to Brand assuming much of the opposition's low-post focus. Guards Cuttino Mobley and Sam Cassell have shown the ability to score in bunches in the past, but they're also a combined 69 years old heading into this season. And we haven't even spoken of Shaun Livingston, once labeled as the team's point guard of the future, who may miss all of the season with a horrific knee injury. Truth be told, much of this season's fortunes hinged on Brand, and without him, a familiar refrain will be heard this year throughout the Staples Center: "Same Old Clippers."
Prior to his injury, Brand was the one player you could count on for reliably big minutes on an otherwise-shaky Clipper unit. Without him, Tim Thomas will be expected to earn his sizable salary and absorb power forward time, while playing upwards of 30 minutes per game. Scorer Corey Maggette will finally earn a starting spot this year, and will see 35 minutes per game at the 3, while Chris Kaman will receive similar time at center. Ruben Patterson will back up Maggette at the 3 and play a little 4, earning 20 minutes per game. Rookie Al Thornton could also see 20 minutes per game backing up the 3 and 4. The reserves down low are uninspiring at best, with Aaron Williams, Paul Davis and Josh Powell all expected to play less than 10 minutes per game.
The starting guard spots will be manned by Sam Cassell (PG) and Cuttino Mobley (SG), though we expect Brevin Knight to gradually cut into Cassell's playing time as the season continues. Mobley will play more than 35 minutes per game while Cassell, at age 37, will average out closer to 25, possibly less if Knight quickly picks up the offense. Knight should average between 20-25 minutes at the point. Quinton Ross should back up the shooting guard spot, and the defensive specialist should earn between 15-20 minutes while playing some 3 as well. Rookie Jared Jordan and free-agent signee Guillermo Diaz should both see less than 10 minutes per game, if that.
Chris Kaman: It will be a sink-or-swim season this year for Kaman, who will not enjoy the freedom of defensive double teams on Elton Brand. Kaman's 2006-07 points (10.1), rebounds (7.8), field goal percentage (45) and free-throw percentage (74) all declined from the previous season, and his blocks (1.5/game) only went up by 0.1. With opposing defenses now free to focus on Kaman, we expect those numbers to further decline, or at least not improve significantly enough to warrant consideration as a front-line fantasy center.
Aaron Williams: Williams has never played enough minutes or produced enough to become a regular fantasy play, and, at age 35, we don't expect that to change much this year. Williams may spell Chris Kaman if Kaman gets into foul trouble, but he will barely see any offensive opportunities.
Paul Davis: Davis has always been a player long on talent but short on production, though the Michigan State product could really surprise should he ever meet his potential. The Clippers could certainly use a player with Davis' size and skill to step up in Elton Brand's absence, but we can't foresee a player who averaged 5.8 minutes per game last year magically becoming a fantasy contributor this season.
Corey Maggette: Maggette assumes the Clippers' scoring load this season with the absence of Elton Brand, and the high-scoring swingman will finally get his opportunity this year to emerge as one of the league's top scorers. Maggette's points-per-48-minutes (26.5) were actually higher than Brand's last year, and he enters this season as the unquestioned starter at small forward. This bodes well for Maggette, as his numbers as a starter last season (19.7 points, 6.4 rebounds and 47% shooting in 31 games) were much higher than his numbers as a reserve (14.9/5.5/43% in 44 games). The only thing that could get in the way of a huge season for Maggette could be his attitude, as he's battled often with head coach Mike Dunleavy and has long been the subject of trade rumors.
Elton Brand: No team faces a greater handicap going into the season than the Clippers, due in large part to the Achilles tear suffered by Brand. Brand, in short, means everything to the Clippers - he led or was near the top of the team in every significant statistical category. That said, his scoring (20.5), rebounds (9.4), blocks (2.2) and minutes (38.4) were all lower in 2006-07 than they were in 2005-06. This year will be much worse, as Brand's best-case scenario will have him returning after the All-Star break, with no guarantee that he'll immediately retain his explosiveness or production. Those considering taking a late-round flier on Brand and stashing him on their disabled list should keep in mind that he won't even be out of a walking boot until late November at the earliest, and that if the Clippers are well out of the playoff race (as they'll almost certainly be), you can be sure they won't rush him back.
Tim Thomas: Big things will be expected out of the Clippers' $24-million-man, as Thomas will be inserted into the starting power forward role until Elton Brand recovers from a torn Achilles. Last year, Thomas missed time with knee, wrist, elbow, back and even gum ailments, while averaging only 11.0 points in the games he did appear in. He shot only 41 percent from the field and 38 percent from three-point range, both well off from his previous season in Phoenix. We expect Thomas' total production to improve this year, if only because he'll get major minutes in Brand's absence. He could be worth a mid-to-late round flier in fantasy drafts and be an excellent source of threes from the power forward spot.
Ruben Patterson: Patterson is coming off his best year as a pro, with 14.7 points, 5.4 rebounds and 2.9 assists while shooting 55 percent from the field for a poor Milwaukee team. We don't expect him to come close to those numbers this year unless Tim Thomas or Corey Maggette gets hurt, and even if he does play major minutes, the 6-5 Patterson may encounter more difficulty against the bigger forwards of the Western Conference.
Al Thornton: Thornton was a volume scorer at Florida State, and was most comparable, ironically, to Maggette while entering the draft. He possesses similar size, quickness and shooting range to Maggette's, albeit with no professional experience. Thornton, like Maggette, also has the tendency to get out of control, and will need to rein in his wild streak before receiving major minutes for the Clippers. If he can quickly absorb the pro game however, we could see him serving as a solid scoring option off the bench for an otherwise scoring-poor Clippers team, possibly more if Maggette is traded.
Cuttino Mobley: Mobley last year had his worst scoring season since 1998-99, and is far removed from his days as one of the league's top scoring guards. At 32, Mobley, does everything decently, but nothing especially well. He has never been much of an assist (2.5) or rebound (3.5) player, and 2006-07 marked the third consecutive season in which his scoring average dropped, this time to 13.8 ppg. His three-point (41%) and free-throw shooting (83%) are two of his strengths, but fantasy owners should expect gradually diminishing returns from Mobley.
Sam Cassell: Cassell started to look his age in 2006-07, starting only 30 games and providing a significant drop-off across-the-board in the games in which he was healthy enough to play. He had nearly a five point dropoff (12.3) from his previous year's production, and Cassell's assists, rebounds, free-throw and shooting percentages also fell off. The Clippers were concerned enough with Cassell that they brought in Brevin Knight to co-occupy the point guard spot, and we expect Knight to eventually receive major minutes at the expense of the 37-year-old former star.
Brevin Knight: The Clippers added Knight to serve as a replacement for injured point guard Shaun Livingston, but their intentions for each were the same: take over the point guard position from Sam Cassell. Knight had an okay year in Charlotte last year (9.1 points, 6.6 assists, 1.5 steals), but he eventually lost playing time to younger point guard Raymond Felton and was deemed expendable. Knight will be playing the Felton role this year, and we fully expect him to take over at the point for the 37-year-old Cassell. Averages of 10 points and eight assists are not out of the question for Knight this year.
Quinton Ross: Ross is strictly a defensive specialist for the Clippers, and holds no fantasy value. The presence of rookie Al Thornton could also eat away at Ross' playing time, and he should not be considered in any fantasy format.
Jared Jordan: The Clippers drafted Jordan in the second round, and many argued that he was one of the best pure point guards in the draft. However, there are huge questions about the Marist product's athleticism, and we expect him to spend this year gaining seasoning and strength in the NBDL.
Guillermo Diaz: Diaz averaged over 20 ppg in the Czech league last season, but the Miami grad should not come close to that this year in the NBA. Expect a fair share of DNP-CD designations, and zero fantasy value from Diaz.
Shaun Livingston: Livingston dislocated his kneecap and tore the ACL, MCL, PCL and lateral meniscus in his knee in late February during one of the more gruesome injuries in basketball history. It would be shocking if he returned this season, and will probably be a shell of himself if he does. Don't count on him for any fantasy production this year.
Corey Maggette: Maggette is now unquestionably the team's top offensive option (if only by default), and will jack up shots this year with even less of a conscience than he usually does. Expect huge offensive numbers from Maggette unless he is traded away to a team with more offensive weapons.
Ruben Patterson: There's a reason that Patterson lasted on the free-agent market until late August, and he probably would have lasted longer than that had Elton Brand not torn his Achilles. Even with Brand out, Patterson will come nowhere close to the career highs he put up on last year's awful Milwaukee team.


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Java Draw Created Nov 06, 2010 460 x 640px Unless noted Copyright 2010 VictoriaRocks.
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i love how this drawing has a burn feel to it :D 5/5!
amazing effect of colours!!! awesome collab !!! 5/5
absolutely stunning! 5h
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Legal Question in Family Law in Mississippi
this is the evidence i have so far: 1. i found them at his place. the door was locked and it took him about a minute to open the door. when he answered the door he was not wearing a shirt and i found her hiding in the bathroom (clothes on). 2. said she went to visit friend out of town. stayed in hotel. on her way back in town her dad was waiting on them and followed her to where she dropped him off at a friends house. 3. i found a letter from him indicating his feelings for her and pictures of him in her night stand with his feelings toward her on the back. 4. had her followed for PI for two weeks. he can verify them being together on four occasions (leaving her car at a friends place and then taking his car back to his place and also her car to his place) each time staying for about 3-4 hours. she uses nothing but her cell phone. she has sold our stock. the check had both our names seperated by the ''&''. i did not sign the check, she forged my name and deposited the check into her savings acct. all of this she thinks i know nothing about except for the letter and pictures.
in your estimation, would this be enough evidence to prove an adultrus affair in the state of Mississippi.
2 Answers from Attorneys
Re: Adultry
Re: Adultry
I would say so.


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Front Office Executive ( 0 - 5 yrs)
Bishnoi Consultancy
RS 1,00,000 To 2,50,000 Dharuhera, Gurgaon, Rewari, Ajmer, Alwar, Bhiwadi
Email, Internet, Calling, Queries, Reception, Records Management
Key Skills : front office executive,back office executive,office assistantce assistant
Electrical Tech Executive ( 0 - 1 yrs)
DSD Global Power Inc
RS 1,00,000 To 1,25,000 Bhiwani, Dharuhera, Gurgaon, Karnal, Rohtak, Haridwar, Kashipur
Electrical Tech Executive (with Three Years Regular Diploma from Govt Institute Only -well Conversant with Solar Electrical Circuit, System & Utilities and Installation/testing . Care & Maintenance of Projects After Installation.
Key Skills : electrical site engineer,electrical supervisor,electrical engineer,senior electrical engineer,assistant electrical engineer,electrical engineer...
Assistant Manager (hr) Dharuhera ( 6 - 10 yrs)
DDS Group
RS 5,00,000 To 6,00,000 Dharuhera, Gurgaon
We are looking for an HR assistant to handle a variety of personnel related administrative duties. You will provide information and clerical support to the HR department and employees regarding human resources activities, policies, processes and proc...
Key Skills : hr assistant manager,hr management,hr assistant,hr manager,hr executive
Crust Infratech Pvt Ltd
RS 2,00,000 To 3,00,000 Dharuhera, Gurgaon, Alwar, Bhiwadi, Neem-Ka-Thana
- Would be responsible for Real Estate Sales in Gurgaon/Bhiwadi/Dharuhera/Neemrana. - Assisting clients for sale of Residential & Commercial Projects. - Visiting the prospects and converting them in to buyers. - Achieve the targeted revenue & prof...
Key Skills : sales executive,real estate sales,sales,sales assistant


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antiquity (definition)
See all of antiquity, no other writeups in this node.
|Roman Empire
|Beethoven Symphony No. 10
|Emperors Decius, Gallus, Aemilianus, Valerian VI
|Hegel's Motivations
|This wicked life
|Gothic Martha Stewart
|Point of Information
|Conduct Towards The Christians From Nero to Constantine
|equo ne credite
|Sonnet LXII
|The Book of the Damned: Chapter 9
|Aristotle's Lost Library, Medieval Andalusia & Chinese Paper, or How Europe Learned to Learn Again and Why the Renaissance Happened When & Where It Did
|Nothing new about Ancient Greece will be learned until the Vatican is plundered
|The Internal Prosperity In The Age Of The Antonines III
|Why Socrates was really executed
|The Case of Charles Dexter Ward
|The Decline and Fall of the Roman Empire
|Sumptuary law
|Hermes Trismegistus
|Unicode Middle Eastern Scripts
|John Adams's 1800 State of the Union Address