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The peptide uroguanylin (Ugn) regulates enteric and renal electrolyte transport. Previous studies have shown that Ugn and its receptor GC-C (a ligand-activated guanylate cyclase) are abundant in the intestine. Less is known about Ugn and GC-C expression in the kidney. Here, we identify a 9.4-kDa polypeptide in rat kidney extracts that appears, based on its biochemical and immunological properties, to be authentic prouroguanylin (proUgn). This propeptide is relatively plentiful in the kidney (∼16% of intestinal levels), whereas its mRNA is marginally present (<1% of intestinal levels), and free Ugn peptide levels are below detection limits (<0.4% of renal proUgn levels). The paucity of preproUgn-encoding mRNA and free Ugn peptide raises the possibility that the kidney might absorb intact proUgn from plasma, where the concentration of propeptide greatly exceeds that of Ugn. However, immunocytochemical analysis reveals that renal proUgn is found exclusively in distal tubular segments, sites previously shown not to accumulate radiolabeled proUgn after intravascular infusions. Thus proUgn appears to be synthesized within the kidney, but the factors that determine its abundance (rates of transcription, translation, processing, and secretion) must be balanced quite differently than in the gut. Surprisingly, we also find negligible expression of GC-C in the rat kidney, a result confirmed both by RT-PCR and by functional assays that measure Ugn-activated cGMP synthesis. Taken together, these data provide evidence for an intrarenal Ugn system that differs from the well-described intestinal system in its regulatory mechanisms and in the receptor targeted by the peptide.
- intestinal natriuretic peptide
- sodium homeostasis
- cGMP
- postprandial natriuresis
- immunohistochemical localization
uroguanylin (Ugn) is a short, cysteine-rich peptide that regulates ion transport in the intestine and the kidney (for a review, see Refs. 18 and 68). Ugn can thus control the movement of electrolytes both into and out of the body and has, accordingly, been proposed to play a homeostatic role in the management of dietary salt. In support of this hypothesis, Ugn knockout mice are hypertensive and excrete orally delivered sodium more slowly than do wild-type animals (10, 49).
The existence of an intrinsic Ugn-based signaling system within the gut is well established. The Ugn gene is strongly expressed in the intestine (2, 11, 45, 55, 56, 62, 88), as is the gene encoding a high affinity, Ugn-activated receptor-guanylate cyclase (rGC) known as GC-C (42, 77, 84). Furthermore, a wealth of evidence confirms that nearly all of the effects of Ugn on intestinal ion fluxes are mediated by GC-C via the downstream effects of cGMP on specific epithelial transporters and channels (6, 15, 22, 25, 29, 35, 51, 65, 71, 72).
In contrast, the renal actions of Ugn are poorly understood. In particular, two fundamentally important questions remain unanswered. The first concerns the tissue source of renally active Ugn. One popular model suggests that renal salt excretion is regulated by Ugn that is released from the intestine (18, 68). In support of this model, biochemical measurements show that the intestine secretes large amounts of prouroguanylin (proUgn) into the plasma (58), and infusion studies with radiolabeled recombinant proUgn demonstrate that the circulating propeptide can be processed within the kidney to Ugn, which is subsequently eliminated in the urine (68). On the other hand, animals consuming high-salt chow appear to maintain normal plasma concentrations of Ugn (24) and proUgn (10), while excreting significantly more Ugn in their urine than do control animals (24). This argues that salt ingestion may mobilize an intrarenal pool of Ugn, rather than a plasma pool supplied by the intestine. Consistent with this alternative hypothesis, several laboratories have detected Ugn-like polypeptides in kidney extracts (58, 62). However, the very low levels of preproUgn mRNA in the kidney (2, 11, 19, 45, 49, 50, 56, 67, 88) have led to the suggestion that Ugn is not synthesized by the kidney, but rather is taken up either from the circulation or from the glomerular filtrate (62), perhaps via receptor-mediated endocytosis (87). Thus the role (if any) of endogenously synthesized Ugn within the kidney remains an open question.
A second area of uncertainty concerns the identity of the receptor(s) and signal transduction mechanism(s) that mediate the natriuretic effects of Ugn in the kidney. The association of Ugn with GC-C and cGMP in the intestine has long been taken as implicit support for the hypothesis that Ugn acts via GC-C and cGMP in the kidney. Direct backing for this idea is provided by evidence that the opossum kidney expresses an mRNA transcript that encodes a GC-C-like receptor (48), as well as the observation that opossum renal and urinary cGMP levels increase after stimulation with Ugn and/or with heat-stable enterotoxin (ST), a bacterial toxin that acts as a high-affinity Ugn mimetic (13, 20, 21). However, expression of GC-C in the kidneys of other mammals is controversial (4, 5, 9, 17, 38, 42, 49, 67, 77, 78, 84), and recent studies have shown that most (although perhaps not all) of the natriuretic actions of Ugn and ST are preserved when the GC-C gene is genetically ablated in mice (3, 4). Thus the roles played by GC-C and cGMP in renal responses to Ugn are not well defined.
Our understanding of Ugn and GC-C expression in the kidney is further complicated by the largely nonoverlapping nature of the experimental techniques and animal species that have been described in previous contributions to the literature. For this reason, we have focused our current studies on a single experimental preparation, the Sprague-Dawley rat, and have used multiple independent techniques (Northern blotting, real-time RT-PCR, quantitative immunoblotting, immunohistochemical analysis, radioreceptor binding assay, and guanylate cyclase activity measurements) to evaluate Ugn and GC-C expression in parallel. Our goal is to establish accurate renal mRNA and polypeptide expression profiles for Ugn and GC-C in the rat model system, where the physiological effects of Ugn on renal salt excretion have been best characterized (1, 16, 17, 58–60, 73, 74).
Sprague-Dawley rats (Charles River Breeding Laboratories, Raleigh, NC) were maintained on a 12:12-h light-dark cycle with free access to water and standard rodent chow. All procedures were approved by the UNC Institutional Animal Care and Use Committee and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Antibodies 6910, 6911, and 6912 were generated by our laboratory, as previously described (64), and antibody 1–11 was generated in the laboratory of Dr. Masamitsu Nakazato. Each antibody was raised against a small (∼15 residue) peptide antigen and therefore targets a specific region of the propeptide, as shown schematically in Fig. 2A. Note that antibodies 6910 and 6911 were raised against the same antigen (PALPLDLQPVCASQE) but in different rabbits, providing an internal check on each other's validity. The two consistently target the same 9.4-kDa proUgn protein on Western blots (58) and produce comparable immunohistological staining patterns. Antibody 6912 (raised against the peptide sequence QQQKSGLLPDVCYNP) also recognizes proUgn robustly on Western blots (58) but is not suitable for immunohistochemistry, as it produces a weak signal that is difficult to distinguish from background labeling. Antibody 1–11 (raised against the peptide sequence TDECELCINVACTGC) has previously been shown to recognize proUgn on Western blots (58), and we show here that it is an effective immunohistological reagent. In addition, in contrast to the other antibodies, it recognizes both proUgn and free Ugn. Antibody 1–11 is available in limited supply and therefore used here exclusively for histological analyses.
Tissue isolation and extraction of protein and mRNA.
Before tissue collection, animals were anesthetized either with urethane (1.6 g /kg body wt ip) or pentobarbital sodium (55 mg/kg body wt ip). For the Northern and Western blot data in Fig. 1, RNA and protein were isolated simultaneously from the same piece of tissue using the TRIzol method (Invitrogen, Carlsbad, CA) as specified by the manufacturer. For the Western blot data in Figs. 2 and 3, mucosal scrapings (obtained from a 2- to 4-cm-length of small bowel located ∼4 cm distal to the pyloric sphincter) or minced kidney tissues were homogenized at a ratio of 4 ml buffer/g tissue in 25 mM HEPES, pH 7.4, containing a protease inhibitor cocktail (Sigma, St. Louis, MO). Homogenates were then centrifuged at 60,000 g for 30 min at 4°C, and the supernatant fraction was collected and stored at −80°C before analysis. For the RT-PCR studies reported in Fig. 8, tissues were rapidly cut into small pieces (∼1 mm3) and immersed overnight at room temperature in RNAlater (Qiagen, Valencia, CA). RNA was subsequently extracted and purified using an RNEasy Mini kit (Qiagen), then treated with DNase I to minimize contamination by genomic DNA.
Western blot procedures and quantitative proUgn assay.
Immunoblots were performed as described previously (45, 58), using antibodies 6910, 6911, 6912, or 1–11 (described above). Immunoblots in Fig. 1B were treated with chemiluminescence reagent (Roche Diagnostics) and exposed to film. For quantitation, films were digitized at a resolution of 600 dpi (LaCie Silverscanner IV) and analyzed densitometrically using NIH Image software (available at http://rsb.info.nih.gov/nih-image/download.html). All other immunoblots were performed with an infrared-emitting secondary antibody (IRDye 800-coupled goat anti-rabbit from Rockland, Gilbertsville, PA, diluted 1:2,000) and analyzed with an Odyssey infrared gel imaging system (LI-COR Biosciences, Lincoln, NE). The quantitative Western blot-based assay is described in detail in a previous publication (58).
Northern blot procedures.
For Northern blots, isolated RNA was denatured by glyoxal, size-fractionated by electrophoresis on 1% agarose gels, pressure-transferred to positively charged nylon membranes (ICN), and allowed to hybridize with an antisense GC-C probe, as previously described (43).
RT-PCR amplifications.
Random hexamer-primed cDNA was prepared from isolated RNA, using the SuperScript III First-Strand Synthesis System (Invitrogen) according to the supplier's protocol. Subsequent RT-PCR analysis employed primer pairs and probes, as specified in Table 1.
Primer sets A and D were used for traditional endpoint RT-PCR. Reactions were performed in a DNA thermal cycler (Ericorp, San Diego, CA), using a GeneAmp DNA amplification kit (PerkinElmer Cetus) and standard ionic conditions. The cycler was programmed to heat to 94°C for 2 min, followed by up to 45 thermal cycles (94°C for 1 min, 60°C for 1 min, 72°C for 2 min), and a final incubation at 72°C for 10 min. PCR products were stained with ethidium bromide, electrophoresed on agarose gels, and visualized under UV light.
Primer sets B and C were used for real-time RT-PCR. Each sample included a fluorescent probe with a reporter dye (fluorescein) at the 5′ end and a quencher dye (tetramethylrhodamine) at the 3′ end, as indicated in Table 1. Amplifications were performed in duplicate in a 96-well plate in the ABI Prism 7700 sequence detector (PE Biosystems, Waltham, MA) for 40 thermal cycles (15 s at 94°C and 1 min at 60°C) as described by Kim et al. (37). Fluorescence was continuously measured in each well, and the fractional cycle at which each sample crossed a fluorescence threshold, CT, was determined using software provided by the manufacturer. CT values were not corrected for differences in amplification efficiency, because efficiencies were consistently very close to the theoretical maximum.
Urinary cGMP measurements.
Rats were anesthetized (55 mg/kg body wt pentobarbital sodium ip) and prepared for renal clearance measurements as previously described (60). Urine was collected via ureteric cannulas, diluted 1:1,000, and assayed for cGMP content by standard RIA (Biomedical Technologies, Stoughton, MA). Samples and standards were acetylated to increase assay specificity and sensitivity.
Guanylate cyclase assays.
Details of this assay are provided in a previous publication (27). In brief, individual kidney subregions (cortex, medulla, and papilla) were hand dissected from tissue slices, using visual landmarks to identify each region. Membranes were isolated by ultracentrifugation, stored on ice, and assayed for activity within 15 min. Protein content was determined on a portion of the diluted membranes using a dye-binding assay (Bio-Rad Bradford Assay). Substrate, cofactors, and a phosphodiesterase inhibitor (IBMX) were added to initiate the reaction at 37°C, with or without a saturating dose of Ugn, ST, or atrial natriuretic peptide (ANP). Reactions were stopped with 6% TCA. Each sample was then extracted with diethyl ether (to remove TCA), and the cGMP content was determined by radioimmunoassay. Results are expressed as picomoles of cGMP synthesized per milligram of protein per minute.
Quantitative ligand binding assay.
This assay is a modification of a previously described receptor binding assay (30). Our version of this assay is based on competitive binding between Ugn and 125I-labeled N-tyrosyl Ugn (Y-Ugn) at GC-C receptors present in isolated T84 cell membranes. Y-Ugn (Bachem Americas, Torrance, CA) was iodinated using precoated iodination tubes according to the manufacturer's instructions (Thermo Scientific, Rockford, IL), and the labeled product was purified by reverse-phase HPLC before use. Membranes were isolated by ultracentrifugation of homogenates of T84 cells and stored at −80°C. Samples and standards were reduced to dryness (Savant SpeedVac, Thermo Electron, Waltham, MA), redissolved in 20 μl assay buffer [HBSS; 20 mM sodium citrate, 0.75% bovine serum albumin (Sigma) at pH 4.5], and incubated for 60 min with T84 cell membranes (1–10 μg protein) and 0.5 nM 125I-Y-Ugn, then filtered through Whatman GF/C glass microfiber filters and rinsed twice with 5 ml of ice-cold buffer (50 mM Tris·HCl at pH 7.4). Bound radioligand was quantified with a gamma counter (Packard Cobra, PerkinElmer Life and Analytical Sciences, Waltham, MA). Ugn concentrations in biological samples were calculated by interpolation on a standard curve, using the equation for a single-site ligand displacement assay where NSB = nonspecific ligand binding (measured in the presence of 10−5 M unlabeled Y-Ugn), Bmax = maximal specific ligand binding, and IC50 = the concentration of competitor that results in 50% displacement of the bound ligand.
Animals were anesthetized with 9:1 ketamine/xylazine (1 ml ip), then perfused with phosphate-buffered saline by cardiac puncture, to clear the kidneys of blood and tubular fluid, followed by perfusion with fixative (4% paraformaldehyde/0.18% picric acid in 0.16 mol/l phosphate buffer, pH 7.2). Kidneys were then removed, postfixed for 2 h in the same fixative, and cryoprotected at 4°C in 0.1 M phosphate buffer, pH 7.2, containing 30% sucrose. Tissue was then embedded in Tissue-Tek Optimal Cutting Temperature Compound (Ted Pella, Redding, CA) and cryostat sectioned at 10 or 14 μm. Some sections were treated with 3% H2O2 for 10 min before immunolabeling to quench endogenous peroxidase activity.
Peroxidase labeling was performed as previously described (47), using a biotinylated anti-rabbit IgG secondary antibody (ABC Elite Kit, Vector Laboratories, Burlingame, CA or Jackson ImmunoResearch Laboratories, West Grove, PA), a streptavidin-biotinylated horseradish peroxidase complex (ABC Elite Kit), and 0.02% DAB (Sigma). Preabsorption controls were carried out with 10 μM synthetic antigen. Tubule counts, tubular diameters, and spatial distributions of tubules and glomeruli were obtained from digitized images by an individual who was unaware of the identity of the primary antibody that had been used to stain each section. Tubular diameters were defined as the diameter of a circular profile, or the length of the short axis of an elliptical profile.
Immunofluorescent double-labeling was performed as described previously (64). Sections were incubated overnight at 4°C with pairs of the following reagents: a 1:50 dilution of mouse anti-CD-26 (dipeptidyl peptidase-4; DPP-IV, Thermo Scientific), a 1:1,000 dilution of rabbit anti-proUgn antibody 6910 or 6911 (raised in-house), a 1:100 dilution of goat anti-aquaporin-2 (Santa Cruz Biotechnology, Santa Cruz, CA), or a 1.66 μg/ml solution of Alexa 658-conjugated peanut agglutinin (PNA; Invitrogen). Sections were then treated as necessary with appropriate secondary reagents at a 1:200 dilution [Texas red donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories), Alexa 568 donkey anti-rabbit IgG (Invitrogen), Alexa 568 goat anti-rabbit IgG (Invitrogen), or Alexa 633 donkey anti-goat IgG (Invitrogen)] and examined under epifluorescence optics. Autofluorescence was evoked by excitation at 488 nm and visualized at 515 nm. Fluorescent images were captured on a Zeiss LSM 510 NLO confocal microscope. Data collected at different wavelengths were stored in individual channels and merged in Adobe Photoshop to produce composite images.
Rat and human Ugn were purchased from Anaspec (Fremont, CA) and Bachem Americas, respectively. Human Ugn exists in two relatively stable isomeric forms (53); we used both isomers for the current studies. Rat Ugn is available only as a fully equilibrated 50:50 mixture of A and B isomers. ST was purchased from Sigma. Rat ANP was purchased from Peninsula Laboratories (Belmont, CA).
Expression of preproUgn mRNA and proUgn polypeptide in the rat kidney.
Northern blot and RT-PCR studies have consistently shown that the kidney contains very small amounts of preproUgn mRNA, relative to expression levels observed in the intestine. It was therefore surprising when significant levels of proUgn were reported in rat kidney extracts (58, 62), suggesting that the preproUgn mRNA-to-protein ratio might vary significantly from tissue to tissue. To examine this quantitatively, we performed side-by-side Northern and Western blot measurements of steady-state preproUgn mRNA and proUgn levels in rat jejunum and kidney.
Relative mRNA expression was evaluated by hybridizing a 32P-labeled antisense Ugn probe against serial dilutions of RNA isolated from each tissue and quantifying probe binding in each sample with a phosphorimager. Regression lines were then fit to the initial linear portions of each dilution series (Fig. 1A). Based on the slopes of these lines, the 600-bp Ugn transcript is ∼100-fold more abundant in jejunal RNA than in renal RNA.
Parallel measurements of polypeptide expression were then performed on Western blots of serially diluted protein extracts, using a propeptide-selective primary antibody (6910). The slopes of the lines fit to the Western blot data (Fig. 1B) show that the 9.4-kDa proUgn molecule is about six times more abundant in the intestine than in the kidney. Thus the data in Fig. 1 identify a substantial mismatch between protein and mRNA expression in the jejunum and kidney, with significantly more proUgn in the latter tissue than would be expected on the basis of its very low preproUgn mRNA levels.
The immunoreactive molecule in kidney is bona fide proUgn.
The high polypeptide-to-RNA ratio in the kidney raises the possibility that the renal Western blot signal in Fig. 1B might represent artifactual antibody binding to a target other than authentic proUgn. To investigate this possibility, we performed side-by-side Western blots with three different proUgn-specific antibodies (6910, 6911, and 6912), each of which was raised independently (see methods and Fig. 2A). In the resulting blots, the immunoreactive renal polypeptide was recognized by all three antibodies and had exactly the same molecular weight as proUgn extracted from the jejunum (Fig. 2B). We then demonstrated that the immunoreactive kidney peptide migrated as a single peak on a C18 reverse-phase HPLC column (Fig. 2C) with a retention time that was identical to that of enteric proUgn (Fig. 2D). Finally, we performed a Western blot with kidney extracts obtained from wild-type and Ugn knockout mice and verified that the putative proUgn polypeptide was observed in the former but not the latter (Fig. 2E). Thus, by multiple criteria, the immunoreactive peptide extracted from the rodent kidney appears to be authentic proUgn. Furthermore, previous studies with recombinant standards have shown that proUgn extracted from the rat small intestine is almost exclusively in full-length, unprocessed form (58). Thus the precise alignment of enteric and renal proUgn in Fig. 2A, and the absence of lower molecular weight processing fragments in the kidney extract, demonstrate that renal proUgn is also stored in fully intact form.
The kidney does not contain detectable levels of stored Ugn.
To investigate whether the renal pool of proUgn is associated with a stored pool of processed Ugn, we used a competitive binding assay to measure the levels of Ugn-like binding activity in rat kidney extracts (see methods). Because the assay can also detect proUgn (very weakly), as well as the structurally related guanylin (Gn) peptide, we used a C18 reverse-phase column to fractionate each extract before performing the assay. We first calibrated the column with synthetic and recombinant rat peptides. Rat Gn eluted in fractions 37 and 38, rat Ugn in fractions 40–43, and rat proUgn in fractions 47–49, as indicated by the black arrowheads in Fig. 3. We then ran a large volume of extract (equivalent to one-third of a kidney) across the column and analyzed fractions 30–65 with the binding assay. No Ugn-like binding activity was detected in fractions 40–43, although a very small peak of activity was present in fraction 38, possibly corresponding to Gn (Fig. 3, ○). We then repeated the experiment with six additional extracts, each derived from an independent kidney. In these experiments we focused exclusively on fractions 39–45 (thus n = 7 for fractions 39–45 in Fig. 3A and n = 1 for all other data points in the figure). No peak corresponding to Ugn was observed in any of these individual column runs. To confirm that Ugn would have been be detected if present in sufficient quantity, we repeated the procedure using a kidney extract that had been spiked with 10 nmol of Ugn standard. Approximately 70% of the added peptide was recovered (Fig. 3, black symbols). Based on this recovery, and the detection limit of the assay, we calculate that a single rat kidney must contain <100 fmol of free Ugn (n = 7).
For comparison, we then measured the propeptide content of the kidney, using a well-validated quantitative Western blot assay (58). After interpolating the observed kidney proUgn values into a standard curve generated with recombinant rat proUgn (Fig. 3B), we found that a single rat kidney contains 25.5 ± 1.4 pmol of propeptide (means ± SE, n = 12).1 Thus, as an upper limit, the free Ugn pool in the rat kidney cannot be >0.4% of the proUgn pool. This is comparable to the situation previously reported for the intestine, where the pool of stored Ugn is also drastically smaller than the pool of stored proUgn (58).
Localization of proUgn to distal tubules in rat kidney cortex.
We then used immunohistological methods to define the cellular distribution of proUgn within the kidney. Because a previous study observed immunostaining in the kidney with an anti-Ugn antibody but not with an anti-proUgn antibody (62), our initial experiments compared antibody 1–11 (an antibody that recognizes both proUgn and free Ugn; see Fig. 2A) with antibody 6910 (a proUgn-selective antibody). Representative immunohistochemical results with 1–11 and 6910 are presented in Fig. 4, B and C (photomontages correspond approximately to the boxed region in Fig. 4A). Both antibodies labeled tubular structures that were scattered throughout the renal cortex (C), whereas labeling was not observed when preimmune serum was substituted for the primary antibody (Fig. 4D), or when the primary antibody was preadsorbed with its corresponding peptide antigen (data not shown).
A few tubular structures were also labeled in superficial regions of the outer medulla. However, medullary labeling dropped off rapidly and was not observed in deeper regions of the outer medulla. The inner medulla and papilla could not be evaluated by immunohistochemical methods, due to nonspecific labeling of most of the cellular structures present in these regions (observed even when sections were stained with preimmune serum or with secondary antibody only). However, Western blots performed on individually hand-dissected layers of the kidney provide direct evidence that these regions do not contain significant amounts of proUgn (Fig. 4E).
The cortical staining produced by antibodies 1–11 and 6910 is shown at higher magnification in Fig. 4, F and G, respectively. As in Fig. 4D, no immunoreactive profiles were observed in nearby sections stained with preimmune serum (Fig. 4H). The labeled structures were clearly identifiable as renal tubules (and distinguishable from glomeruli and blood vessels) on the basis of their sizes and characteristic morphologies.
Further analysis revealed that the populations of tubules labeled by each antibody were identical in terms of their mean diameters (Supplementary Fig. S1a; all supplementary material for this article is available on the journal web site), relative abundances (Supplementary Fig. S1b), and distributions within the cortex (Supplementary Fig. S1c), arguing strongly that both antibodies were targeting the same population of tubules. This, coupled with the failure to observe detectable levels of free Ugn in kidney extracts (Fig. 3), provides convincing evidence against the previously reported conclusion that the renal tubules contain Ugn but not proUgn (62).
To better define the subcellular localization of the immunoreactive material within the tubules, we examined horseradish peroxidase-labeled preparations at higher magnification with differential interference contrast optics. A representative tubular profile stained with antibody 6910 is shown in Fig. 5A. The immunoreactive material is obviously intracellular and is distributed relatively evenly throughout the cytoplasm, within and beneath an apical zone that is occupied by nuclei. This same pattern (apical nuclei and cytoplasmic immunostaining) was also evident in brightfield images of immunolabeled sections that had been lightly counterstained with hematoxylin (Fig. 5B). Thus the renal pool of proUgn does not represent filtered material that had adsorbed to luminal surfaces of the tubular epithelium.
Proximal tubules can be distinguished from distal tubules on the basis of 1) their selective expression of brush-border enzymes (such as CD 26, also known as dipeptidyl peptidase IV) (86); and 2) their intense autofluorescence (generated by a population of acid phosphatase-containing granules that is highly enriched in the cytoplasm of the proximal tubular cells) (54). To investigate whether proximal tubules contain proUgn, we double-labeled a section of kidney cortex with an anti-CD 26 antibody (red) and the anti-proUgn antibody 6910 (blue). In the merged image, the two staining patterns were completely nonoverlapping (Fig. 5C and Table 2), arguing that proUgn is not expressed in the proximal tubule. We then compared the distribution of proUgn to the endogenous autofluorescence and observed that proUgn-expressing tubules (blue) were unequivocally distinct from the strongly autofluorescent profiles (green), as shown both for antibody 6910 in Fig. 5D and for antibody 6911 in Fig. 5E. Thus, by two criteria (lack of CD 26 expression and lack of strong autofluorescence), the proUgn-immunopositive tubules appear to be distal rather than proximal (Table 2).
To further confirm a distal pattern of expression, we compared the labeling obtained with antibody 6911 to that obtained with PNA, a lectin that binds to a carbohydrate motif that is highly expressed in distal tubules, as well as in the brush borders of the earliest portions of the proximal tubule (61). As shown in Fig. 5F, PNA labeled two morphologically distinct structures in sections of kidney cortex: a prominent set of ring-shaped profiles whose associated staining appears as a sharply defined pair of parallel lines (4 examples are marked by white arrowheads), and a much less abundant set of profiles whose diffuse staining is located in close proximity to the tubular lumen and resembles the brush-border staining that we had observed with the anti-CD26 antibody (2 examples are marked by yellow arrowheads). By comparing Fig. 5F to the corresponding autofluorescence image in Fig. 5G, it is evident that the former structures were only marginally autofluorescent (thus corresponding to distal tubules) while the latter structures were strongly autofluorescent (thus corresponding to proximal tubules). Figure 5H shows the distribution of proUgn-positive tubules in the same section. The PNA-positive profiles marked with white arrowheads (distal) were immunopositive for proUgn, whereas those marked with the yellow arrowheads (proximal) did not stain for proUgn. As seen in the merged image (Fig. 5I), every tubule in the section that can be identified as distal [i.e., PNA-positive (red) but not strongly autofluorescent (very faintly green)] was positive for proUgn whereas every tubule that can be identified as proximal (i.e., bright green, due to strong autofluorescence) was negative for proUgn. These correlations were confirmed in every double-labeled section that we examined. Thus proUgn is expressed only in distal tubules and appears to be present in all distal tubules.
The distal components of the nephron that reside in the cortex include the distal convoluted tubules, the connecting tubules, and the cortical collecting ducts. The connecting tubules and cortical collecting ducts express aquaporin-2 (AQP2), while the distal convoluted tubules do not (14). Figure 5J shows AQP2 staining in the kidney cortex (red profiles). Every single AQP2-positive tubule was also immunoreactive for proUgn (blue profiles), as can be seen by comparing Fig. 5J with Fig. 5L, or by examining the merged image in Fig. 5M (for reference, 2 AQP2-positive, proUgn-positive tubules are marked by white arrowheads). We consistently observed an identical colabeling pattern in numerous tissue sections. The distal identity of the AQP2-positive tubules was verified by their lack of autofluorescence (white arrowheads in Fig. 5, K and M). The observation that every AQP2-positive tubule also stained positively for proUgn indicates that the propeptide is expressed in both AQP2-positive nephron segments (the connecting tubules and the cortical collecting ducts). In addition, however, a number of proUgn-positive tubules were also AQP2 negative (2 examples are marked by the yellow arrowheads). These AQP2-negative tubules are also distal (because they were nonautofluorescent; Fig. 5, K and M) and therefore must correspond to the distal convoluted tubules.
Taken together, the immunohistochemical data presented in Fig. 5, C–M (and summarized in Table 2) indicate that proUgn is broadly expressed throughout the distal nephron, whereas it is completely absent from the proximal components of the nephron.
GC-C (enteric Ugn receptor) is not appreciably expressed in the rat kidney.
Although it is universally acknowledged that GC-C mRNA is abundant in the intestine, only some studies have reported its presence in the kidney (4, 5, 17, 38, 67, 78), while other studies support the opposite conclusion (9, 42, 49, 77, 84). Interpretation of these published data is further complicated by differences in the probes, primers, techniques, and animal species that have been employed by different laboratories. Here, we have used two different primer pairs (Table 1) to evaluate GC-C expression in the Sprague-Dawley rat, using both traditional endpoint RT-PCR (primer set A) and quantitative real-time RT-PCR (primer set B).
Figure 6A shows endpoint RT-PCR results. When we analyzed RNA isolated from proximal jejunum, a well-defined amplicon of the appropriate size (340 bp) appeared at cycle 27 and increased in abundance during subsequent cycles until it reached a plateau level at around cycle 40. No such PCR product was evident if the RNA sample was not reverse transcribed to cDNA, even after 45 cycles (data not shown). By contrast, when we analyzed kidney RNA, the only observable amplification products were low-molecular-weight primer dimers and a diffuse background that increased over time but did not resolve into an individual amplicon. To assess the quality and integrity of the input RNA used for these experiments, the same intestinal and renal samples were tested with primers specific for rat cyclophilin A, a housekeeping gene that is consistently expressed in most tissues. In both samples, an appropriately sized amplicon appeared at cycle 17 and increased to a plateau by cycle 27, suggesting that mRNA degradation was not a significant problem in either case.
To measure GC-C expression more quantitatively, we then developed primers and fluorogenic probes for real-time RT-PCR analysis. Using this method, we found that cycle threshold (CT) values for β-actin, another commonly measured housekeeping gene, were very similar for RNA isolated from the jejunum and kidney (Fig. 6B, gray bars), while CT values for GC-C diverged widely between the two tissues (Fig. 6B, white bars). The average CT for GC-C was close to that of β-actin in the small intestine but very much higher than that of β-actin in the kidney. Indeed, the CT value plotted for the kidney is an underestimation, as 18 of 23 amplification reactions did not reach threshold within 40 cycles (when the PCR reaction was terminated) and were therefore assigned nominal CT values of 40. GC-C expression was then normalized to β-actin expression for each tissue by determining ΔCT (defined as CT of β-actin − CT of GC-C for each sample), and relative expression levels were calculated as 2−ΔCT (Fig. 6B, black bars). Consistent with the more qualitative data presented in Fig. 6A, rat intestine shows abundant GC-C expression, while rat kidney shows exceedingly low expression that, in most cases, fell below the limit of detection.
Does Ugn regulate cGMP metabolism in the rat kidney?
The failure to detect significant renal expression of GC-C mRNA (Fig. 6) raises the possibility that the natriuretic actions of Ugn in the rat kidney are cGMP independent. However, it is also possible that Ugn does affect cGMP metabolism but does so by acting through a signal-transducing guanylate cyclase that is distinct from GC-C. Therefore, to directly investigate the effect of Ugn on renal cGMP metabolism, we measured urinary excretion of cGMP before, during, and after intravascular infusion of Ugn. This well-established procedure has been used as a sensitive way to detect coupling of other natriuretic agents, such as ANP or nitric oxide, to the cGMP second messenger cascade (28). Indeed, our initial control experiments demonstrated a rapid and robust increase in urinary cGMP levels during intravascular infusion of ANP at a dose of 7 nmol·h−1·kg body wt−1 (Fig. 7A, white symbols, n = 2).
We then examined the effect of Ugn in the same experimental setting. For these experiments, we used human Ugn, which is available in two isomeric forms. The A isomer is a potent ligand for GC-C and produces large cGMP increases in GC-C-expressing cells, whereas the B isomer has no known effect on cGMP metabolism in any target cell, although it is a very effective natriuretic agent in the rat kidney (60). As might be expected from its lack of effect on cGMP metabolism in other systems, Ugn B did not stimulate urinary cGMP excretion by the rat kidney when infused at 50 nmol·h−1·kg body wt−1 (Fig. 7A, gray symbols, n = 3), a dose that evokes maximal natriuretic activity in rats. Perhaps less predictably, Ugn A also did not stimulate urinary cGMP excretion when delivered at 25 (Fig. 7A, black symbols, n = 3), 50 (data not shown, n = 5), or 200 nmol·h−1·kg body wt−1 (data not shown, n = 1). These doses span the range over which infused human Ugn A produces maximal natriuretic responses in rats (60). The physiological efficacy of the peptides thus argues that their failure to evoke cGMP production does not reflect a failure to reach appropriate receptors. Furthermore, previous studies have documented substantial recovery of human UgnA and B in the urine after intravascular infusion (60), confirming that both peptides would have the opportunity to interact with luminally oriented receptors at any point along the nephron.
It has also been reported that infused proUgn is natriuretic (58, 59), presumably because the propeptide is converted to Ugn and other metabolites within the renal tubules (68). Interestingly, a recent study (59) has shown that, mole-for-mole, proUgn is a more potent natriuretic agent than Ugn, suggesting that intrarenal processing of proUgn may produce an alternate metabolite that is more active than Ugn. To test whether proUgn (and/or any of its metabolites) might activate cGMP synthesis, we measured urinary cGMP levels before, during, and after infusion of proUgn at a natriuretic dose of 10 nmol·h−1·kg body wt−1. No change in cGMP excretion was evoked by the infusion (Fig. 7B, black symbols). We also measured Ugn levels in the same urine samples and observed a large increase in Ugn excretion during and after proUgn infusion (Fig. 7B, white symbols), confirming that processing of the propeptide had occurred in these experiments.
Thus the data in Fig. 7, A and B, are consistent with the idea that, unlike ANP, neither Ugn nor proUgn regulates renal sodium excretion in a cGMP-dependent manner. One caveat to this interpretation, however, is that cGMP might have been produced in response to peptide infusions but was retained in an impermeable intracellular compartment and/or broken down by a phosphodiesterase before it could reach the urine. To test this, we performed standard ligand-activated guanylate cyclase assays with broken cell preparations, in the presence of IBMX, a broad-spectrum phosphodiesterase inhibitor. We prepared membranes for these assays from three different regions of the rat kidney (cortex, medulla, or papilla) to increase the likelihood that we would observe a response even if it was restricted to a specific anatomic region. We also used rat Ugn (a spontaneously equilibrating 50:50 mixture of A and B isomers) to avoid possible complications that might arise from applying the human peptide to rat receptors. Under these conditions (Fig. 7C), we could still observe a prominent stimulatory effect of ANP (10−6 M), which was most evident with membranes prepared from isolated papillas. However, neither rat Ugn (10−6 M), nor its more potent analog ST (100 U/ml), had any effect on cyclase activity in membranes derived from any region of the kidney.
Our studies provide a comprehensive description of Ugn and GC-C expression in the kidney of the Sprague-Dawley rat. The principal conclusions are as follows. 1) The rat kidney contains significant amounts of authentic proUgn (∼16% of the level found in the intestine; Fig. 1B), despite expressing only very low amounts of preproUgn mRNA (∼1% of the level found in the intestine; Fig. 1A). 2) Using a sensitive and selective binding assay, we failed to detect Ugn itself in extracts of rat kidney (Fig. 3). Based on the limit of detection of the assay, the pool of free Ugn maintained by kidney (if any) is <0.4% of the pool of proUgn. Thus very little of the proUgn stored within the kidney is processed intracellularly to mature Ugn. 3) The relatively abundant intrarenal proUgn pool is localized primarily to the kidney cortex (Fig. 4 and Supplementary Fig. S1c), where it is restricted to distal convoluted tubules, connecting tubules, and cortical collecting ducts (Fig. 5 and Table 2). 4) GC-C, the previously identified Ugn receptor, is abundant in the intestine, but nearly undetectable in the kidney at either the mRNA level (based on RT-PCR analysis; Fig. 6) or the functional level (based on measurements of Ugn-evoked cGMP synthesis; Fig. 7). Thus, consistent with many previous studies, these observations support the well-established hypothesis that Ugn signaling in the kidney involves a receptor that is distinct from GC-C (3, 4, 60, 79, 80, 81).
Renal expression of proUgn.
Ugn was originally discovered in urine (32, 40). A potential tissue source was subsequently defined when Ugn was purified and sequenced from intestinal extracts (31, 33, 45). This biochemical evidence for enteric production of Ugn has since been corroborated and extended by numerous Northern blot studies showing high and relatively selective expression of preproUgn mRNA in the intestine (2, 11, 19, 45, 49, 55–57, 67, 88). However, several of these Northern blot studies have also noted the presence of very low levels of preproUgn mRNA in the kidney, an observation that has been consistently confirmed by more sensitive RT-PCR assays (5, 13, 36, 50). Here, we show that these low levels of renal mRNA are associated with an unexpectedly high abundance of proUgn polypeptide.
The reason for the divergent polypeptide-to-mRNA ratios in the kidney and intestine is unclear. Possibilities include 1) an inherently high rate of proUgn synthesis in the kidney, relative to that of the intestine; 2) comparable transcription and translation rates in the two tissues, but a higher rate of propeptide processing, secretion, and/or degradation in the intestine; or 3) a renal transport mechanism that translocates previously synthesized propeptide from the plasma (where it is plentiful) (58) into the kidney, with no need for significant local transcription. Convincing evidence against the latter of these hypotheses has been provided by a study that traced the metabolic fate of radiolabeled recombinant proUgn after intravascular infusion (68). Although these experiments did reveal significant accumulation of radioactivity in the kidney, the retained material was almost entirely in the form of free amino acids, rather than intact proUgn, and was associated with proximal tubules, rather than with the distal tubular profiles that we have currently identified as the site of endogenous renal proUgn expression (Fig. 5). Thus circulating propeptide does not appear to contribute to the pool of proUgn that resides in the distal tubules.
ProUgn localization.
Our identification of the distal tubule as the site of proUgn expression in the rat kidney can be compared with previous renal localization studies, in which Ugn-like immunoreactivity has been reported in proximal tubules (23), distal tubules (62), and an unidentified tubular compartment that is restricted to the corticomedullary border (7, 85). Our results are in best agreement with the analysis of Nakazato et al. (62), who identified the distal convoluted tubule as the site of expression of Ugn. However, the immunostaining observed in the Nakazato study was obtained with an antibody directed against the mature Ugn peptide, whereas the propeptide-selective antibody 6912 failed to produce a detectable signal in their experiments. For this reason, the authors concluded that the kidney contains free Ugn, but little or no proUgn, a conclusion that contrasts strikingly with the data that we present in Figs. 1–5.
There are several likely explanations for the immunohistochemical discrepancy between our results and those of Nakazato et al. (62). First, we fixed our kidneys with Zamboni's fixative, which includes both paraformaldehyde and picric acid, whereas the Nakazato study employed a formaldehyde/phosphate buffer-based fixative. In previous analyses of enteric proGn and proUgn expression, we have found that the antigenicity of the propeptides was much better preserved with Zamboni's fixative than with fixatives that lack picric acid (46, 64). Second, our experiments focused on anti-proUgn antibodies 6910 and 6911, whereas the Nakazato experiments focused on anti-proUgn antibody 6912. In our hands, the latter is a relatively ineffective histochemical reagent, producing weak signals in the intestine and nearly undetectable signals in the kidney (data not shown).
The localization of authentic proUgn to distal tubules raises intriguing issues about Ugn signaling. 1) Most importantly, the kidney must now be recognized as a second major tissue reservoir for the propeptide, presumably operating in parallel with the small intestine. This introduces the possibility that the abundant urinary pool of Ugn (12, 40) could be derived from renal proUgn, from enteric proUgn, or from propeptide supplied by both tissues. Indeed, each tissue source could play a distinct role in regulating whole body sodium balance, as is the case for other endocrine agents that are generated both extra- and intrarenally [such as angiotensin (41) and ANP/urodilatin (34)].
2) The kidney does not secrete proUgn into the circulation (58). Thus any proUgn released from cells in the distal nephron would likely be delivered directly to the tubular lumen. Furthermore, since proUgn cannot be detected in normal rat urine (62) (59), any such luminal propeptide must be converted to Ugn before excretion. Most of the currently recognized luminally active proteases in the kidney are restricted to the proximal tubule (26) and thus upstream from the site of renal proUgn expression. Nevertheless, the distal nephron contains several membrane-associated ectoproteases that could potentially serve as luminal processing enzymes (63, 66, 83). In addition, a broad spectrum of soluble proteases has been identified in urine (69), implying that these enzymes are also present in the luminal fluid within the distal nephron. Thus mechanisms are available that could potentially generate Ugn from distally secreted proUgn.
3) A distal site of proUgn expression also implies a distal site of physiological action. Natriuretic agents that inhibit sodium reabsorption late in the nephron (such as ANP) typically evoke a natriuresis without a significant kaliuresis (70). This is consistent with the previously described saliuretic effects of infused proUgn in the anesthetized rat, where prominent increases in total and fractional sodium excretion were observed with either marginal increases (58) or net decreases (59) in total and fractional excretion of potassium. However, unlike ANP, which acts in the collecting duct exclusively through a cGMP-dependent mechanism (39), our current experiments reveal that the effects of Ugn are strikingly independent of cGMP (Fig. 7).
GC-C expression in the rat kidney.
The failure to implicate cGMP in the renal actions of Ugn is somewhat surprising, given that Ugn has long been linked to intestinal cGMP metabolism through the catalytic activity of its well-known enteric receptor, GC-C (18). However, the mouse kidney responds to Ugn after genetic ablation of the GC-C gene (3, 4, 80), and the rat kidney displays robust responses to Ugn (4, 16, 17, 59, 60, 74) despite its failure to express detectable levels of GC-C mRNA or protein in RT-PCR assays (Fig. 6), functional assays (Fig. 7), or Western blot assays (67). Thus it appears that the rodent kidney contains an alternate Ugn receptor that mediates the effects of the peptide in the absence of GC-C. Further corroboration of this hypothesis is provided by reports of GC-C-independent responses to Ugn in rat kidneys (60), isolated mouse cortical collecting ducts (80), isolated human cortical collecting ducts (78), and a human kidney epithelial cell line (81), as well as by the observation that GC-C knockout mice excrete orally delivered sodium normally, in contrast to the delayed sodium excretion and hypertension that characterize Ugn knockout mice, presumably because the kidneys of the GC-C-deficient animals respond to Ugn via this alternate receptor (10).
Despite this abundant evidence that GC-C is not required to support renal responses to Ugn, it must be acknowledged that several previous RT-PCR studies have reported GC-C expression in the rat kidney (5, 17). We are not sure why these results differ from ours. However, we do note that GC-C expression has proven to be relatively malleable. Thus GC-C mRNA is ectopically expressed in the rat liver during fetal development (42), as well as during postinjury hepatic regeneration (52, 76). Expression is also observed in esophageal adenocarcinoma cells (but not normal esophageal epithelium) and is further upregulated in these neoplastic cells after exposure to bile acids (8). In addition, GC-C expression shows a pronounced diurnal rhythm in the rat intestine (75) and is affected by oral salt intake in both the kidney (17) and intestine (44). Thus some of the variability in renal GC-C expression that has been reported in the literature may reflect differences in the handling, feeding, or harvesting of experimental animals. Indeed, even in our own study, 5 of the 24 rats included in our real-time RT-PCR analysis showed presumptive evidence for a small amount of renal GC-C expression (although the levels in these animals were still exceedingly low relative to the levels in the intestine). Thus, although GC-C is undoubtedly not the main receptor that mediates the natriuretic effects of Ugn in the rat kidney, it may regulate an as-yet undefined component of renal function in some animals under some conditions.
We have presented evidence that the rat kidney contains unexpectedly large amounts of proUgn, relative to its strikingly low levels of preproUgn mRNA. Thus the propeptide is relatively abundant, and apparently selectively expressed, in the tissues that control the flow of sodium into and out of the body (the intestine and the kidney, respectively). Like the intestine, the kidney does not process its intracellularly stored pool of propeptide before secretion but presumably releases it intact for subsequent extracellular processing. Given the restricted expression of proUgn in the distal nephron, and the previous observation that the kidney makes little or no contribution to the circulating plasma pool of propeptide (58), we believe that the most likely processing site for renal proUgn is within the tubular lumen at a distal site. However, this hypothesis has not been confirmed, and the protease(s) that would act at this location (and the processing products that they might generate) have not yet been identified. The receptor that mediates the natriuretic effects of Ugn or proUgn within the kidney is also not known, although our data strongly support the idea that it is not GC-C, and does not employ a cGMP-dependent signaling mechanism. Intriguingly, this raises the likelihood that such a novel receptor would display a ligand specificity that is distinct from that of GC-C. In this regard, we have recently found that intravascularly infused proUgn actually evokes a more effective natriuresis than an equivalent dose of intravascularly infused Ugn (59), arguing that proUgn is converted within the kidney to an as-yet unidentified natriuretic peptide that is distinct from, and more potent than, Ugn. Thus the Ugn signaling pathways in the gut and kidney appear to have diverged significantly with respect to the receptors, the ligands, and the propeptide processing mechanisms that are active in each tissue.
This work was supported by the National Institutes of Health (HL078980) and the American Heart Association (0755397U).
No conflicts of interest, financial or otherwise, are declared by the authors.
The authors thank Christopher Cazzolla for performing the double-blind morphometric analyses, James Anderson for generously providing the AQP2 antibody, Mitchell Cohen for very kindly supplying us with Ugn knockout mice, and Kathleen Dunlap, Bill Arendshorst, and Carie Facemire for helpful comments on the manuscript.
Present address of X. Qian: Dept. of Physiology, Univ. of South Alabama, Mobile, AL 36608.
↵1 Note that this value is lower than a previously published estimate of renal proUgn levels from our laboratory (58). We feel that the current determination is more accurate for two reasons: relative to the earlier study 1) the kidneys in the present study were more rigorously cleared of plasma (which contains very high levels of propeptide), and 2) the recombinant proUgn standard in current use is of higher purity, and its concentration was determined by a more accurate method.
- Copyright © 2011 the American Physiological Society
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text:
More Great News for Gingrich in Iowa
There are now two more polls, in addition to the two polls released last weekend, showing that Newt Gingrich has the lead in the Iowa Caucus. The PPP poll finds Gingrich with a nine point lead over second place Ron Paul. From PPP:
Newt Gingrich has taken the lead in PPP's newest poll of Iowa Republican caucus voters with 27% to 18% for Ron Paul, 16% for Mitt Romney, 13% for Michele Bachmann, 9% for Rick Perry, 6% for Rick Santorum, 4% for Jon Huntsman, and 1% for Gary Johnson.
Gingrich has gained 19 points since PPP's last poll of the race in early October. Also showing momentum are Paul whose support is up 8% and Bachmann whose support is up 5%. Romney has dropped 6 points since then with the other candidates mostly standing in place.
The Washington Post-ABC News poll finds that Gingrich has a 15 point lead in Iowa among likely caucus goers. From ABC News:
Given these views, 33 percent of likely Republican caucus-goers currently favor Gingrich for the GOP nomination, with 18 percent apiece for Romney and Paul. Rick Perry garners 11 percent support; Michele Bachmann, 8; Rick Santorum, 7; and Jon Huntsman, 2 percent.
In addition to these two polls, a third poll from CBS News/New York Times has even more good news for Gingrich. The poll finds that Republicans in Iowa believe Gingrich is the most electable of the nominees, barely edging out Romney. From CBS News:
In a CBS News/New York Times poll released Tuesday, 31 percent of likely Iowa caucus-goers said Gingrich had the best chance among the current GOP field to defeat President Barack Obama in 2012. Twenty-nine percent said Romney had the best chance.
All other candidates polled in the single-digits on the electability question: Texas Gov. Rick Perry with 9 percent, Rep. Ron Paul with 6 percent, Rep. Michele Bachmann with 3 percent, and both John Huntsman and Rick Santorum with 1 percent.
The electability argument is one of the best arguments Mitt Romney's campaign has. If the GOP base actually thinks Gingrich stands a better chance of winning a general election, Romney could be in real trouble and not just in Iowa.
Things are looking surprisingly good for Gingrich right now.

subset: commoncrawl


text:
Staff at a business on the North Lynn Industrial Estate have been evacuated.
Two fire crews from the Lynn station have been called to a site in Bergen Way after a smell of fire was reported at 9.10am today (Wednesday, February 12).
The fire service say there is no obvious signs of fire.


text:
Harry Carey, Jr. (Henry George Carey, Jr.), (born May 16, 1921, Saugus, Calif.—died Dec. 27, 2012, Santa Barbara, Calif.), American actor who brought an aura of youthful naivete and likability to a series of classic movie westerns directed by John Ford, many of them starring John Wayne. Carey was featured in some of Ford's most acclaimed films, including 3 Godfathers (1948), She Wore a Yellow Ribbon (1949), Rio Grande (1950), Wagon Master (1950), The Long Gray Line (1955), The Searchers (1956), and Two Rode Together (1961). He was the son of famed silent western star Harry Carey, who appeared in more than 260 movies between 1909 and his death in 1947, and Olive Fuller Golden Carey, who played more than 75 supporting roles, some of them in her son's films. The younger Carey got his big break in director Howard Hawks's western Red River (1948), which featured his father and starred Wayne. In addition to his many movies, Carey made numerous television appearances, notably in a recurring role as the camp counselor in the mid-1950s Walt Disney series Spin and Marty; his final part was in the made-for-TV movie Last Stand at Saber River (1997). Carey's autobiography, Company of Heroes: My Life as an Actor in the John Ford Stock Company, was published in 1994.
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text:
iOS devices get some Turtle Beach love
After I saw Turtle Beach's plans for Xbox One headsets during my time at E3, I was shown their iSeries line. The iSeries, as you've probably guessed just from the 'i' in the name, is Turtle Beach's line of headsets perfect for Apple users and their products -- iMac, iPad and iPhone.
The most basic of the three iSeries headsets I was shown was the i10. While the three headsets share the same features -- sleek design, all white and silver like iOS devices, and a little red Turtle Beach symbol -- the i10 is the simplest of the three. While quality isn't spared, the i10 lacks the features that the other two have, and the reason is to keep costs down and offer different choices for Apple users with different budgets.
And so, the i10 provides a wired option for Apple users. To keep the sleek and stylish design, the i10 uses dual hidden microphones and a special algorithm to pick up voice patterns without the use of a boom or in-line mic. The battery lasts up to 15 hours and is rechargeable while in-use with a USB cable. The best feature of the headset -- a feature that's present in all three of the headsets -- is an Active Noise Cancellation (ANC) button that toggles the headset between blocking out outside noise and letting you hear it. It's an awesome little feature that'll be really useful when trying to block out the sounds of your friends and family while you watch a movie, listen to music or play a game.
If you prefer a wireless option to the wired i10, then Turtle Beach has you covered with the i30 headset. It has the same battery, Active Noise Cancellation, hidden microphones and sound quality as the i10 headset; the only difference is that it uses Bluetooth 4.0 to pair with any Bluetooth-enabled devices -- iPhone, iPad or iPod Touch -- to listen to your media or take phone calls.
Continue to PAGE 2 to see the i60 headset.


text:
Deteriorating underground sewage system of Tema Community 4 is causing enormous health problems to the residents of Residents at Tema Community 4.
A mixture of feaces ,urine and dirty water has been spilling and running into resident's homes instead of gushing out into the sea as portions of the pipes have cracked.
Major outbreak of diseases such as Cholera is feared if the problem is not solved.
Several residents complained to Joy News' Joseph Opoku Gakpo when he visited the area.
One of the residents said, "even last week I took her to the hospital, my little girl. She was vomiting having high temperature we went and they treated her for malaria. She can't stand the smell. I think they said mild asthma."
Another resident complained, "it is an eyesore the fecal matters are showing, the sewage central pumping unit are threatening to blast. You can hear roaring waters."
Others complained, the sewage is causing cracks in the buildings.
"You have seen the sewage is razing buildings down ,buildings are cracked right down from the foundation level all caused by the sewage…..If urgent measures are not taken, houses would be razed down people within Tema Central will fall sick and die."
The Tema MCE Isaac Ashai Odamten noted, his outfit was aware of the stench, sewage spill and residents discomfort.
"We have actually assessed the situation of the sewage problem in the whole metropolis, we have identified key flash points in Community 2 and 4 and parts of Community 1 and 8 while the other places do have some challenges but the flash points is where we have hinted."
He explained although Tema Community Four had s "weak sewage infrastructure" the attitude of residents has further worsened the problem.
He explained, "properties have been built on the sewer line" and this is not allowing the maintenance work to be done."
Nonetheless, the Assembly, he said, had made some progress in improving the situation.
"We have changed some pipes , rods and now it is flowing better…it is has improved," he stated.
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5 Years : From Nov 2012 to Nov 2017
LONDON--Royal Dutch Shell PLC (RDSA.LN) said Monday that it purchased for cancellation 784,000 Class B shares at 2122.25 pence per share.
-Class B shares closed Monday at 2,136 pence valuing the company at GBP133.7 billion.
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Category: Education
Date: 04.07.2013
Time: 10.56
Our project is about electricity
Today I have set up our stand
The most amazing thing is the other schools stands!
Barnqack schools stand was really good as it had hand made examples. The things on the stand were also really good.
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text:
Not unlike any other politician, Republican presidential challenger Mitt Romney can use every vote that he can get.
As the events of last week's Republican National Convention amply demonstrate, Romney and his party are reaching out to demographic groups — independents and moderates, single women, blacks, Hispanics, "the youth vote" — whose members ordinarily lean more heavily toward Democrats. But, interestingly, there is one group that currently resides within the Republican Party that they are in danger of losing.
That group consists of Ron Paul supporters.
And what an energized, enthusiastic bunch it is.
The only problem for Romney and his running mate, Paul Ryan, is that these Paul supporters aren't in the least enthusiastic for the GOP ticket. In fact, they are reaching — or have already reached — the verge of voting for a third party candidate.
Ironically, the Texas Congressman's devotees, though encompassing all age groups, are comprised predominantly of young voters — i.e. exactly that demographic that the Republican Party is laboring indefatigably to attract.
Yet these voters and other Paul constituents seem poised to jump the GOP ship — a move, they say, that their own party has provoked them to make.
For one, the party establishment caused quite a ruckus among not just Paul's supporters but its base when it decided not only to disenfranchise some of Ron Paul's delegates but also to rewrite delegate rules, effectively diminishing Ron Paul's influence at last week's convention or the influence of any other candidate not favored by the establishment at future conventions.
In other words, Paul supporters are miffed at the GOP for the same reason that radio talk show hosts Rush Limbaugh, Mark Levin, and others were miffed: In rewriting its rules, the GOP establishment succeeded in suppressing those voices — Paul supporters, Tea Partiers, social conservatives — within its ranks that threatened its objective to woo moderates.
Secondly, although Paul was apparently offered an opportunity to speak at the convention, the offer had conditions attached. The speech would have to be approved by the convention planners, but, just as importantly, it would include an unqualified endorsement of Romney on Paul's part.
The retiring Paul passed on it.
Finally, while the RNC honored Paul with a video tribute, this decision generated controversy among establishment Republicans, leading the establishment conservative movement's flagship publication National Review to publish a denunciation of the video. In an article appearing in its on-line edition (National Review Online), "The Problem with Paul," Jamie M. Fly and Evan Moore write that it was "a mistake" to honor Paul.
Referencing the truncated convention schedule, the authors lament what they perceive to have been a "missed ... opportunity" for the convention planners "to reverse the ridiculous and regrettable decision made by the Romney campaign to feature a video tribute" to Paul.
Fly and Moore concede that because of the number of delegates that Paul won, and because of his supporters' propensity to be particularly "vocal," it is not unreasonable that the Romney campaign should strive to avoid giving offense. "Concessions have already been made to them [Paul supporters] on extraneous issues during the drafting of the platform," and "a speaking slot has been given to" Paul's "son, Senator Rand Paul."However, Paul's critics conclude, "paying tribute to Representative Paul is a step too far."
Fly and Moore, as if to disabuse Paul and his supporters of all doubts regarding their fellow partisans' feelings toward them, add that "instead of honoring Paul on the way out, the delegates in Tampa should be cheering his departure." They explain that Paul "has left a legacy of extremism and falsehoods that need to be driven from the party, not embraced by it."
"It's important to remember how far outside the mainstream Paul and many of his supporters are," the authors continue. The views of Paul on which the authors set their sights, as Paul's supporters and their opponents have by now come to expect, pertain to foreign, not domestic, policy.
It is Paul's foreign policy vision that has elicited the ire of the Republican Party and its spokespersons in the media (like National Review Online). Fly and Moore are incensed specifically about Paul's position on the issue of Iran.
Paul accuses the GOP of once again "beating the war drums" in its rhetoric regarding a nuclear-armed Iran. Neither the CIA nor the IAEA, according to Paul, have said that Iran is on the verge of obtaining a nuclear weapon, and under the rules of the Non-Proliferation Treaty, Iran is permitted to have nuclear weapons anyhow. He ridicules the degree to which Republicans particularly remain "obsessed with Iran and the idea that Iran is a threat to our national security."
Fly and Moore criticize Paul for allegedly painting "a picture of a peaceful and benevolent Islamic Republic that has never actually existed." They also refer to his argument as an "apologia for the ayatollahs" and judge it to be "as absurd as it is dangerous." Furthermore, they contend, "it is wholly irresponsible for anyone who aspires to national leadership" to take the position that Paul takes.
Paul's objectors also allude to his "trail of similar factual errors and conspiracy-mongering on issues ranging from the defense budget to America's position overseas, the wars in Afghanistan and Iraq, and even the origins of the attacks of September 11, 2001."
Fly's and Moore's charge to the contrary notwithstanding, it has now been years that poll after poll has shown that Paul's views on these foreign policy-related matters are very much within the mainstream. Moreover, inasmuch as Republicans persist in their belief that the wars in Iraq and Afghanistan are worthwhile endeavors, that our interventionist foreign policy had nothing to do with the terrorist attacks of 9/11, and that we should remain on course with interventions in Iran, it is they who appear to be "far outside the mainstream."
In 2006 and 2008, Americans voted Republicans out of power in overwhelming numbers. President George W. Bush retired from his second term with an approval rating hovering at about 30 percent. While multiple factors doubtless accounted for their fatigue with the GOP, key among them was their weariness with what an ever growing number of Americans came to see as two regretful, avoidable wars in the Middle East.
Current polls establish that they have not changed their minds on this score, for foreign policy registers low among their concerns.
If this year's RNC was any indication, none of this is in dispute: The 2012 Republican National Convention was the first such convention in 60 years that excluded all explicit references to war.
It would be premature to conclude that Ron Paul's promise that his movement would one day "become the tent" of the Republican Party is now coming to fruition. In any case, though, his influence is felt.
Romney, not unlike any other politician, may want to place some stock in that.


text:
Dragon's Dogma
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- Dragon's Dogma
- Rent and Be Rented Redux #23
YOU ARE LIMITED TO LISTING TWO GTS. YOU ARE REQUIRED TO RENT AND RETURN TWO PAWNS FROM THE LIST EACH WEEK. The next week will end October 11, 2013. You may join at any time; I will be fair if you join close to the deadline.
The Rent List is updated in response to GAMEFAQS PMs that are sent to my attention. The nice thing about Googledocs is that the link is HERE:
Refresh it frequently, it changes as often as several times daily.
1. A posted Thank You is required in a specific format for each time you are rented. This is how I track rents for who is included on the next list. An example:
Thanks are due to:
Dorkmeister; 123,456 RC; 5/5/5*; Sour Ambrosial Meat
NOTE THE TWO BOLD SEMI-COLON SEPARATORS! Listing the rating, gift and a comment is optional, the first two fields are not. If you miss some of this information because you haven't learned how to check information in your rentals, that's ok… we'll teach you how to do that.
2. If you do not rent from the group, your name/GT will not show up in the Thank You posts. This is grounds for removal from the next list. I will list the non-active GTs prior to doing so and give at least 24 hours notice via a topic post called "The Naughty List." If sufficient thank-yous are posted in the following 24 hours, you're all good.
It is not to be construed as a Walk of Shame—the Naughty List is a courtesy warning, not necessarily impending doom. PM me with your concerns and we'll talk.
3. This is not the place to list your name and then never be heard from again. The group expects EVERYONE to be an active renter and post received thanks, too! Yes, the Rent Topic is a chatty one, you need not participate in the conversation. Posting your rent thanks is sufficient.
Rent whomever you like. There should be no need to pay RC for any pawn in the Rent Group, feel free to send someone a Friend Request. Unless the person's list is full, I highly doubt it will be refused.
4. CONSTRUCTIVE CRITICISM. We are all gamers here, and we all want our pawns to improve. If you have a suggestion for someone's pawn, make it. Recipients-- take advantage of the advice being offered, there are thousands of hours of experience available here. Do not take it personally if you receive advice you weren't looking for, as advice is subjective. Do not take it personally if your advice is not taken, as advice is subjective.
Questions about the game/pawns/leveling are considered on topic.
5. This is a very active topic. The post limit is 500, if it's filled and closed another one will be started.
6. Be excellent to each other. Gifts are nice, but gifting is totally up to the discretion of the Renter and they are not an expectation. Trolling will not be tolerated; you will be immediately dropped/shunned if your behavior is unacceptable and/or odious. Personality conflicts are easily avoided by simply being polite.
GT/PSN: Karathrax, Alt: Sekhmeti
Want in?
Send me a PM with your GT, pawn's name, level and vocation.... as simple as that.
GT/PSN: Karathrax, Alt: Sekhmeti
TheRicFactor 4 years ago#3
XBL / PSN: TheRicFactor
Beat me to it, slow editing the date I guess
GT: cmStone Pawn: Kurgan
GT: cmStoned Pawn: Jenassa (lvl 70's)
EdgeofLethe 4 years ago#5
@Mick, Fenix, and Lonerwolf, thanks for taking a look! I think I'm going to get to play some today!
I forgot about Longevity being on there, so I will swap that out. Also, I think I'll try one set of barbed nails for a while, haven't experimented with KD much.
Re: Fearful Din. I find it very useful until the Everfall, and then it takes a hit to usefulness. Mostly it's great crowd control for bandits, goblins, saurians, whatever light mobs you encounter in big groups until the latter stages of the game. Especially since my arisen is always right in the fray, suddenly having the whole crowd stunned can make things really easy. Also, it sounds bad as hell =p!
It is the mark of an educated mind to be able to entertain a thought without accepting it - Aristotle
TheRicFactor 4 years ago#6
Draken is feeling FABULOUS!
EDIT: Apologies to Mick, Kurgan jumped off the back of a Poisoned Undead and into a pit. Re-renting at next Riftstone.
XBL / PSN: TheRicFactor
killenTime01 4 years ago#7
Welcome back Doc
GT/ KillenTime 01 Pawn Elvira
GT/ KillenTime 02 Pawn Amber
Kurgan jumped off the back of a Poisoned Undead and into a pit.
He's been known to do that, no worries. He rode one off the bridge to the black abby once. Just as i was going back up the tower to the riftstone he comes running around the corner with a smirk on his face. Musta just caught the ledge below. Hopefully he's not embarrassing himself too much :)
Blackmail shots of Draken, always good for a laugh.
GT: cmStone Pawn: Kurgan
GT: cmStoned Pawn: Jenassa (lvl 70's)
TheRicFactor 4 years ago#9
Mick65 posted...
Kurgan jumped off the back of a Poisoned Undead and into a pit.
He's been doing great. I see what you mean though about Hokino not healing him when Exhilaration kicks in though. I noticed that in a fight against a Firedrake. Draken and Kurgan were both critical and she actually healed Draken instead of Kurgan. The only thing I notice with Kurgan is he sure does run through his Stamina in most fights. He usually ends up huffing and puffing at least once. I guess I'm dragging he fights on too long. He's still doing great though. I'm thinking of on one of my next runs, grabbing Kurgan, my Fighter, Rebecca, and chaning Hokino to Fighter and watching them all wreck monsters. Hokino actually has a lot of experience as a fighter. I'm thinking it might be pretty fun.
XBL / PSN: TheRicFactor
@ Mick can you look at Yamino in the rift this is the closest to the 2nd closest to my desired look, ill show you the most desirable look after u look at this one, as there is a huge flaw in it i cant look over. As of armor i just want debilitation resistance i know there is a ring that increases all resistances however i do not possess it : / tell me what ya think
XBL: XxReaperxX 007 | Pawn Yamino Hikari 200 Fighter
GoLD3nH3r0 | Pawn Kage-Hikari <150 Fighter.... Yes i DO like fighter...
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- Rent and Be Rented Redux #23
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[Apple TV 2] Video (rendering?) quality - Printable Version
+- Kodi Community Forum (https://forum.kodi.tv)
+-- Forum: Support (https://forum.kodi.tv/forumdisplay.php?fid=33)
+--- Forum: General Support (https://forum.kodi.tv/forumdisplay.php?fid=111)
+---- Forum: iOS (https://forum.kodi.tv/forumdisplay.php?fid=137)
+---- Thread: [Apple TV 2] Video (rendering?) quality (/showthread.php?tid=130909)
Video (rendering?) quality - ronk - 2012-05-07
New user here (of this forum, of XBMC, and of the ATV)
Recently moved to ATV+XBMC, very impressed with the software!
Got a few problems and questions, but with a bit of searching in this forum, got the thing
to work quite beautifully - streaming through WIFI from an NFS share, no problems with 720p and
even above, having a blast
One thing which I wasn't able to solve in spite of searching, is the an issue with picture quality
when viewing videos.
Everything looks fine except - the colors. They look like the video is very compressed, it's very
noticeable in (what's supposed to be) smooth gradients on large surfaces - the color transitions look like
a very compressed jpeg picture (see the "Before" side in this image to get what I mean)
This problem does not appear when viewing the same videos on the same TV with the same connection, when
using an XTreamer.
I read in this forum about a problem that existed before in the ATV port's implementation of "rendering", but got
the impression that it was supposed to be fixed by now (in Eden).
Any help would be much appreciated!
My setup information:
XBMC (11.0 Git:20120331-ebfd899), Platform: Darwin iOS (11.0.0 AppleTV2,1, Version 5.1 (Build 9B179b)). Built on Apr 1 2012
The full debug log can be seen here: http://xbmclogs.com/show.php?id=2328
EDIT - here is another debug log which contains messages from playing a MKV file and a couple of streamed TED talks.
Absolutely nothing was installed on the ATV2 except for the JailBreak and XBMC.
RE: Video (rendering?) quality - Ned Scott - 2012-05-08
I know at least one or two other users had this issue (though most don't seem to), and we figured out something... but it escapes me right now. I'll see if I can pull up a link, unless someone else beats me to it.
RE: Video (rendering?) quality - ronk - 2012-05-08
(2012-05-08, 07:15)Ned Scott Wrote: I know at least one or two other users had this issue (though most don't seem to), and we figured out something... but it escapes me right now. I'll see if I can pull up a link, unless someone else beats me to it.
Well, I found this and this, but there is no mention of what the solution was, if any.
The previously-solved problem is described here - seems to have had much the same symptoms.
Here is another mention, with screen captures that show the issue.
HTH, really looking forward to have this sorted out.. Thank you


text:
Joined: 24 February 2007
Posts: 12300
|Sriti Jha: Hina Khan is super cute and sweet
|Though Sriti Jha is not new to the small screen, she is a new member in the Star Parivaar. She has entered the parivaar with her show, 'Shaurya aur Suhani'. As a result, she has been nominated in the 'Favorite Naya Sadasya (female)' category.
"It feels really great" exclaims Sriti "I am more excited because I'll be performing also, however that's what I know as of now. Other things have not been decided. But frankly speaking more than the performance, I am happier about the fact that I am now part of the Star Family."
She's very thrilled about being associated with Star loves playing Suhani on her show. She says, "It feels really great when you get to play really interesting characters like Suhani. I am very thrilled to be a part of the grand Star Pariwaar. I am a new member of the family and that's the reason I've been nominated for this."
Surprisingly, Sriti will vote for Akshara in the Favourite Naya Sadasya (female) category. She tells us, "I truly think that the most deserving person for this is Akshara as in Hina. In fact I am voting for her only.(Laughs) There are three main reasons for this: the first one is that her character is very good, she's very popular and most importantly, she works in Ye Rishta which is the number 1 show presently."
She says she likes Ye Rishta so much that she follows it whenever she gets time. She says, "I feel that Hina is super cute. I've just met her briefly once but she's damn sweet. I even follow her show and I think she's very talented!"
Apart from Sriti, other people who have been nominated in the Favourite Naya Sadasya (female) category are Hina Khan (Akshara of Ye Rishta), Shivshakti (Bebo of Sabki Laadli Bebo), Parakh Madan (Krishna in Burey Bhi Hum Bhale Bhi Hum) and Pooja Bose (Vrinda in Tujh Sang).
link to the article : http://www.starplus.in/spa09/newsdetails.aspx?nid=10
The following 3 member(s) liked the above post:
-Imu.M-, taibaaa, sofiaaax.,
Joined: 21 July 2008
Posts: 1921
Joined: 25 June 2006
Posts: 103971
Joined: 05 October 2008
Posts: 1088
Joined: 29 July 2008
Posts: 18534
|Last Post
-!- Sudha/Devika (Sriti) Appreciation Thread -!-
Author: -Sharona- Replies: 89 Views: 15209
|27 May 2010 at 9:07am by .Vrish.
when did sriti ?
Author: Bluebird10 Replies: 7 Views: 963
|27 September 2009 at 3:43am by Bluebird10
Sriti is amazing
Author: -Yuks- Replies: 12 Views: 1465
|24 September 2009 at 1:31pm by Miss_Random
Jyoti's Sriti Jha in jail! - Article
Author: niveditha Replies: 1 Views: 1381
|04 September 2009 at 1:42am by .Vrish.
Sriti Jha 's pics on IF
Author: swanithak Replies: 3 Views: 4653
|16 August 2009 at 12:14pm by swanithak
And finally this actor will play the lead role of Ranjit Singh..
From second/third leads to now becoming the main leads... These actors ...
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Looking for some filmi fun? Guess who this stunner is!
Know your Bollywood stars well? Test your filmi gyaan here!
This multi-faceted personality has been around for a while but has been spotted in films only sparingly until a recent item song put her on the map again.
Apart from her dancing skills, this hottie is also known as a songstress.
Know who she is?
Please click NEXT to find out!
Image: Guess who!


text:
PAS' Yuhaizad Abdullah and Umno's Roslan Ahmad will face off for the next seven days in Merlimau.
MERLIMAU: The Merlimau by-election will see a straight fight between Yuhaizad Abdullah (PAS) and Roslam Ahmad (Umno) after they have successfully filed their nominations at the Politeknik Merlimau here this morning.
Three objections were raised by PAS but were subsequently dismissed. At about 11.20am, both Yuhaizad and Roslan were declared the official candidates.
Newcomer Yuhaizad, 38, is the Malacca PAS Youth deputy chief as well as Jasin PAS division chief.
Meeting the press later, Yuhaizad merely smiled and was vague when answering questions on his campaign.
"I will provide more details at a later date but what I can promise now is that I will go down to the ground to meet everyone," he said.
Roslan heads Perbadanan Melaka Holdings, a state corporation.
"I am confident of a win because of the support and cooperation of the BN machinery nationwide," he said.
Roslan's father, Ahmad Ithnin, was the former Sungai Rambai assemblyman . When asked whether this would boost his chances in Merlimau, he said, "I have my own set of capabilities but yes my father will add an advantage to my campaign."
Better advantage
Among the notable faces in the PAS camp were PKR president Dr Wan Azizah Wan Ismail, Selangor Menteri Besar Khalid Ibrahim, Petaling Jaya Utara MP Tony Pua, PAS deputy president Nasaruddin Md Isa, Selangor exco Xavier Jeyakumar, Batu MP Tian Chua and PKR vice-president N Surendran.
Spotted in the BN camp were MIC president G Palanivel, Deputy Education Minister Wee Ka Siong, Defence Minister Zahid Hamidi and Perak Menteri Besar Zambry Kadir.
The Merlimau seat fell vacant following the death of Umno assemblyman Mohammad Hidhir Abu Hassan on Jan 20. Hidhir wrested the seat from PAS in 2008 with a majority of 2,154 votes. This by-election will be the 15th since the 2008 general election and the first in Malacca.
Malays form the majority of voters there and pundits say 80% are loyal Umno supporters. The Merlimau seat has been touted as an easy win in this BN stronghold but the ruling government is not letting its guard down.
"BN will have a better advantage," said Zambry. "But we've learnt not to take anything for granted. The candidate must take it seriously to assure the voters of his capability."[youtube]-40oQ-x2w4M[/youtube]
Palanivel took a bolder stand in declaring that the Indian community is safely in BN's pocket.
"Seven of 10 Indians will vote BN," he said confidently. "The other three won't turn up. My target is to achieve a bigger majority than the 70% achieved in Tenang (Johor)."
The opposition, though well aware of the difficult road ahead, is determined to give BN a tough fight.
"Galas (Kelantan) and Tenang have not broken our spirit," said Nasruddin. "Our candidate's advantage is that he is a local and will be able carry out his duties easily here."
"Besides, even (former prime minister) Dr Mahathir Mohamad has lost to PAS in Kedah before. So we will give it our best shot…"
Basic issues
Meanwhile, ousted former Perak menteri besar Nizar Jamaluddin shot down talk of PAS facing opposition among the non-Malays, saying his engagement with the Chinese voters yesterday assured him of their support for the opposition.
Pua said DAP would further strengthen this support by explaining how Pakatan's policies are able to address their basic issues.
Besides the candidates and politicians, another "personality" was showered with attention by the media and supporters.
Kiki, a three-year-old male cat, was spotted lounging in the shade clad in BN "attire".
"He is a staunch supporter," his owner, Suraya Abdul Hakim, said proudly.


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Live blogging – Accessible Technology – Gila Gertel
Monday, April 7, 2008
The Hilton hotel, 17:30 (which is now), Gila Gertel from the Israeli Accessibility Organization is giving a very interesting lecture about accessible technology. Do come and listen, it will open your eyes. Gila Gertel is getting ready for the lecture. It Started Now Not to many people came. Maybe 10-15. This is actually quite sad for me, as this is a topic which is very close to my heart. Gila starts with some background about accessibility - Who needs it? How many? and so on and so forth. ...
no comments


text:
I'm stumped! Our '96 chevy 1500WT w/4.3L V-6 (5-speed manual/air/2WD). With 170K miles seems to have a mind of its own. It doesn't have cruise control, BUT. Often (NOT ALWAYS, though!) You take your foot off the gas and the truck keeps going along at the same speed! Disengage the clutch, and the engine RPM stays at 1500-2000RPM until (and here's the catch.) We come to a COMPLETE STOP! The engine then drops right down to a stable idle of about 800 RPM as soon as the wheels stop turning!
I have removed, but not replaced the IAC valve. It looks like new and does not stick at all. This happens in any weather, engine cold or hot, and there are no engine codes set. This has been going on for 4 years and 50K miles with no change. Any ideas?
Thanks in advance.
have the same problem?
Sunday, April 15th, 2007 AT 10:45 AM


text: Any particular reason why Bass Central would tell me over the phone that all Fenders will be going up in price in a couple of days? I called regarding a 57RI Fender P and a fellow named "Grasshopper" made me a "one time only offer" of $1349, down from the $1499 they have it posted it at. He said he was offering the deal because "in a few days Fenders are going up." I stated how I knew the AVRI line was being revamped with a higher price tag of $1999, which he didn't seem to know about. I asked if he was sure that it was 'all' Fenders, as most had already recently taken a price hike and he said yes, ALL Fenders. If this is true, that stinks, but if its not, wouldn't that be a bit of a sleazy sales tactic? (most 57'RI's were being blown out at pretty much every other retailer for close to $1100). Either one of the nations most respected, premier bass retailers is hitting me with real good offer, is misinformed or is giving me a deal under the pretense that a price hike is going to happen. I like to think that they are just misinformed, but after speaking with my Sweetwater rep immediately after, he told me that as far as he knew, aside from the new AVRIs, Fenders prices had already gone up and they don't anticipate any more hikes in the immediate future? Anyone know the real story?


text:
This product is no longer available.
Pale Blossom Beaded Pashmina Shawl
This pashmina is made with 70% cashmere and 30% silk. Has 3" beaded tassels on both ends. Measures 36" x 80".
This product is no longer listed on FindGift.com by this retailer. Check the Similar Gifts section above to find other gifts that may be suitable.


text:
The question isn't specific, so it allows a wide-ranging response. Ill take it as an invitation to look at unstructured data domains in three senses of the term: data types and sources (obviously), deep information content (very important), and challenges posed by two of the much-talked-about big data Vs, variety and velocity (the third being volume).
More than text
Text is the most software-accessible and analytically useful form of unstructured data, but unstructured data comprises much more than just text. Electronically stored images, audio, and video are similarly unstructured. To a computer, a piece of content in these data domains is typically just a mass of bits. Descriptive metadata, minimally capturing file type and encoding, provides enough information to support content display and editing, but Photoshop, iTunes, and the Chrome Web browser dont have a clue about the meaning of the information they render.
To extract and exploit meaning -- parsing facts and opinions from a written sentence, recognizing a face in an image, detecting emotion in a recorded voice -- you need analytical technologies. Text analytics, particularly natural-language processing of text, is far more advanced than analogous techniques applied to images, audio, and video. Our first set of much-more-work-needed data domains, then, consists of nontextual forms of unstructured data.
What information do we seek in these sources?
- Images: We wish to determine "features" that include people, places, and objects, along with image context and in-image interrelationships (e.g., President Obama is delivering the 2012 State of the Union address, with the vice president and House speaker in the background). We also would like to infer information about the image-capture apparatus.
- Audio: We wish to determine who or what is heard in an audio stream; what activities, speech, emotions, etc. are captured; what interrelationships occur among the things recorded; and how the sound producers and their relationships change over the course of the recording. Speech is an audio special case. It can be treated as spoken text with extra-textual information, such as emotion inferred from tone, pace, and volume.
- Video: This type of information is the cross-product of the information content of images and audio.
Were nowhere close to doing a great job in these unstructured data domains. (And maybe, in a few years, well be recording smells, tastes, and other sensations as unstructured data.) Work is still needed on text, too.
Going deep
Much more work is needed to go deep and get at the rich information content of unstructured sources. We focus a lot on the entities named in source materials (people, companies, locations, etc.) and not enough on facts, events, relationships, and fine-grain attributes such as sentiment and identity. More attention is going to opinions, attitudes, and emotions (and they are subject matter for my upcoming Sentiment Analysis Symposium), which can be derived from text but also from speech and images.
In addition, much more work is needed on targeting genre (whether the material is explaining, informing, arguing, etc.), identity (what language use and style, or the captured physical characteristics, tell us about someones age, sex, and cultural or economic background), and narrative (how topics and attitudes change in the course of a multiple-participant exchange).
Lastly, putting aside volume, the two other big data Vs that define unstructured data domains -- velocity and variety -- need much more work.
Right time, right way
Data velocity refers to high rates of data production and transmission. Competitive pressure is making the ability to transform and analyze high-velocity data "in flight," whether in real-time or "right-time," into a big data imperative. Applications include online commerce, customer service and support, reputation management, and news-influenced automated trading.
Handling variety is similarly important. Enterprises need access to a spectrum of social, online, and enterprise data sources, and they need to handle (acquire, store, analyze, and disseminate) data of all types, including the unstructured sorts Ive cited. You also need to be able to link data across sources and types the right way.
So we have a right-time velocity imperative that extends to as-produced unstructured data, and we have a right-way imperative: integrated, not siloed.
Lets put a spin on the "right way" concept as it relates to data domains, unstructured or otherwise. Our analytical tools have been doing only half the job. Weve been collecting and crunching data and delivering insights via interfaces. Yet dashboards, tables, and visualizations are often not the best way, or the right way, to deliver information. Take this article. Its a written narrative. The panel I attended yesterday conveyed information via spoken presentations and conversations, along with presentation slides and nonverbal speaker gestures and expressions, all of which are "unstructured" in a machine sense but readily understandable by us humans given the linguistic, cultural models we unconsciously apply to understand one another.
Another "right way" -- an unstructured data domain where much more work is needed -- involves natural-language generation by machines and more natural interaction of all "unstructured" sorts. Much more work is needed, indeed!