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The Greatest Story Ever Told(1965)The life of Jesus Christ. For more about The Greatest Story Ever Told and the The Greatest Story Ever Told Blu-ray release, see the The Greatest Story Ever Told Blu-ray Review published by Jeffrey Kauffman on March 31, 2011 where this Blu-ray release scored 3.0 out of 5. Director: George Stevens Writers: James Lee Barrett, George Stevens Starring: Max von Sydow, Dorothy McGuire, Robert Loggia, Claude Rains, José Ferrer, Charlton Heston » See full cast & crew The Greatest Story Ever Told Blu-ray, Video QualityIt's never a good sign when a film comes with the infamous "best available elements" disclaimer, and unfortunately that's very much the case with The Greatest Story Ever Told on Blu-ray. An Ultra Panavision 70mm film would seem to be the perfect source material for a stunning high definition transfer, and while this AVC encoded 1080p presentation in 2.75:1 is most certainly a decided step up from the previous SD-DVD, it is still littered with defects and artifacts that keep it from being anywhere near reference quality. On the plus side, colors are often gorgeously saturated and usually very robust, and color timing appears accurate. Fine detail is good to very good in close-ups but diminishes to murkiness in midrange and far range shots, and in fact a lot of this film looks quite soft. While contrast is generally good, some of the darker scenes (and there are a lot of them in this film) suffer from moderate crush with an attendant lack of differentiation between foreground and background objects. The worst thing about this transfer, aside from damage to the master itself, is abundant flicker, registration differences and a lot of artifacting, including low grade noise, shimmer, aliasing and moiré. Grain is largely intact but in some of the opticals becomes dangerously close to noise levels (a good example is in the Crucifixion scene, where the cloudy background comes perilously close to completely devolving). At least we have an anamorphically enhanced transfer in the correct aspect ratio, so the opening and closing titles are at last at least relatively legible. The Greatest Story Ever Told Blu-ray, Audio QualityMuch better is the lossless DTS-HD Master Audio 5.1 mix, especially with regard to one of Alfred Newman's crowning achievements as a film composer. This was one of Newman's favorite scores, and it's easy to see (hear?) why. This is haunting, lushly orchestrated music (Newman had a great team assisting him, including his usual co-hort Ken Darby, as well as Hugo Friedhofer, Fred Steiner, Leo Shuken and Jack Hayes), and it sounds magnificent with the brilliant fidelity of this lossless track. Dialogue for the most part sounds just fine, though it's quite evident (even more so than on the DVD) how much of this film was post-dubbed, with really noticeable differences in hall ambience for the looped work. There's consistent but never really very overwhelming immersion on the 5.1 track. This was obviously meant to be a reverential piece, and as such, aside from the score and some crowd scenes, there simply isn't that much opportunity to exploit a surround mix. There are one or two commanding bursts of LFE, notably the huge thunderclap that caps Jesus' ascent into Heaven in the crucifixion scene. The Greatest Story Ever Told: Other Editions
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I found this article to be very interesting in detailing the incredible challenges facing our 911 dispatchers.
Category Archives: Uncategorized - Page 3
DC 911 Mobile Calls
Digital Homestead Records
Heard this story on NPR Radio yesterday afternoon "Digital Homestead Records Reopen A Crucial Chapter Of U.S. History" – http://www.npr.org/2014/07/02/327797193/digital-homestead-records-reopen-a-crucial-chapter-of-u-s-history.
The Homestead Records Project seeks to digitize the over 800,000 Homestead Records from nearly 200 land offices in all 30 Homesteading States. Nebraska records were the first to be digitized, and they are now complete. Next up is Arizona, followed by Illinois, Indiana, Iowa, Ohio, Nevada, and Alaska (in that order), but for now the records for the remaining 29 states are only available in hard copy in the National Archives – http://www.nps.gov/home/historyculture/track-the-progress-of-the-homestead-records-project.htm.
http://www.nps.gov/home/historyculture/homesteadrecords.htm – NE Records are currently available on – http://www.fold3.com/title_650/homestead_records_ne/, but fold3.com is not a free site. This could be an interesting historical set of land records to geocode to our state GIS maps when they become available [and if they are free].
Posted for Phil Worrall, Executive Director, Indiana Geographic Information Council, Inc.
Mapping 'Big Data'
How old is your house?
Friday afternoon considerations
How do you spend your time?
Mapping our Energy System
This is a great series of maps showing what our energy infrastructure looks like.
Northwest Group Acquired by Hexagon AB
On Wednesday, Hexagon AB announced that it had acquired Northwest Geomatics, Ltd. (aka Northwest Group) which is a longtime NSGIC Platinum Sponsor. Tim Crago (Vice President, Northwest Group) briefed NSGIC's leadership on the acquisition and reaffirmed his commitment to sponsoring NSGIC. He also alluded to exciting new product offerings, resulting from the acquisition, that he hopes to announce at the NSGIC Annual Conference in Charleston, South Carolina. Stay tuned…
Esri's Contribution to ConnectED
Daniel Edelson is vice president for education at the National Geographic Society and directs National Geographic's Center for Geo-Education. He recently posted an interesting article on their Education Blog that describes the value of Esri's commitment in support of President Obama's ConnectED Initiative. Esri will provide free access to ArcGIS Online to all elementary and secondary schools in the United States. Follow this link to learn more about this initiative. The Huffington Post published a related article titled "The New Space Race" by Thomas Fisher, Dean of the College of Design, at the University of Minnesota. The Association of American Geographers also published an article in it's newsletter about Esri's donation to this important program.
Smithsonian Article on History of GIS
In case you haven't seen this interesting article:
Gerrymander, anyone?
Mother's Day data
The Economic Potential of Geospatial Data
While I'm preaching to the choir, this op-ed makes some good arguments to share with others. I especially like the point at the end, that GIS is poised to generate the next transformation of data online.
How healthy is your county?
NSGIC Address Program Survey 2013
In October 2013, the NSGIC Address Working Group conducted a survey to describe the level of activity of states and territories with address location databases and programs that support them. Address program points of contact had previously been identified via NSGIC state and territory liaisons.
The results of the survey were presented at the NSGIC 2013 Annual Conference in Kansas City. In addition, state-specific slides were made available for comparisons in a deck and via the NSGIC State Information sub-site. Responses were also made available for download in spreadsheet form for further analysis at will.
What was most remarkable was the level of response. 49 of 57 states and territories expressed whether they had programs for or plans to develop an address dataset for their jurisdiction. 19 of these admitted to no formal program, however at least half of these reported a high level of interest or activities in the planning or informal implementation stages. And none of these responders selected "No Interest/No Demand" as a reason for not administering an addressing program. Lack of personnel, funding, or coordination were instead listed as the primary reasons.
Of the 30 states and territories reporting a program, most reported representations of over 80% of their population or jurisdictions. Their profiles of activity show that address data aggregation was the principle role of states and territories, although coordination, distribution, standardization, quality assurance, technical assistance and standards promulgation were also common roles. Less than five states or territories indicated that they were address assigners.
911 Dispatch, Emergency Management, Broadband Mapping, and Enterprise Geocoding needs were the most common drivers among those states and territories reporting addressing programs, each with differing weights for both use and contributions. Funding for these programs ,as reported by 17 of the 30 states administering address programs, is predominantly supported via federal grants and telephone service charges. The costs for start-up and maintenance reported varied widely.
Data collection from local jurisdictions is predominantly via lower technological means, specifically email, File Transfer Protocol (FTP) servers or other Managed File Transfers (MFT) services, or on physical discs, although collection via database and web service access may be growing. Distribution after collection was primarily reported to be via address-matching (geocoding) services, FTP/MFT, web map services, and static data download websites. Data is largely being stored in spatial relational databases.
For collection and re-distribution of address data, 40% of the respondents reported no formal agreements were necessary AND their data was made available to the public, while agreements to make data available to state and/or federal governments were still common for others.
The use of internal or State standards was most common. The use of national address standards (FGDC, NENA, USPS) were significant but not predominantly used in quality assessments. Rather general aspects of attribute and spatial accuracy and database normalization or integrity constraints were common among them.
The data for at least a quarter of those reporting addressing programs is field verified, and likely over half of the data contains "subaddresses" or additional location information most often indicating in which building and at which unit an address is located. Many address datasets describe at what feature (building, driveway, parcel) the address point is being located.
For more information regarding the survey, please contact Russell Provost or Nathan Lowry. Those who are interested in this topic more broadly are invited to join the Address Committee by contacting Russell Provost.
Sincerely, Nathan Lowry
GIS Outreach Coordinator
601 East 18th Avenue, Suite 220, Desk D-23, Denver, CO 80203-1494
email@example.com | www.colorado.gov/oit
How am I doing? Please contact my manager Jon Gottsegen (email@example.com) for comments or questions.

subset: commoncrawl


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Transient receptor potential canonical (TRPC) channels are non-selective cation channels involved in receptor-mediated calcium signaling in diverse cells and tissues. The canonical transient receptor potential 6 (TRPC6) has been implicated in several pathological processes, including focal segmental glomerulosclerosis (FSGS), cardiac hypertrophy, and pulmonary hypertension. The two large cytoplasmic segments of the cation channel play a critical role in the proper regulation of channel activity, and are involved in several protein-protein interactions.
Here we report that SNF8, a component of the endosomal sorting complex for transport-II (ESCRT-II) complex, interacts with TRPC6. The interaction was initially observed in a yeast two-hybrid screen using the amino-terminal cytoplasmic domain of TRPC6 as bait, and confirmed by co-immunoprecipitation from eukaryotic cell extracts. The amino-terminal 107 amino acids are necessary and sufficient for the interaction. Overexpression of SNF8 enhances both wild-type and gain-of-function mutant TRPC6-mediated whole-cell currents in HEK293T cells. Furthermore, activation of NFAT-mediated transcription by gain-of-function mutants is enhanced by overexpression of SNF8, and partially inhibited by RNAi mediated knockdown of SNF8. Although the ESCRT-II complex functions in the endocytosis and lysosomal degradation of transmembrane proteins, SNF8 overexpression does not alter the amount of TRPC6 present on the cell surface.
SNF8 is novel binding partner of TRPC6, binding to the amino-terminal cytoplasmic domain of the channel. Modulating SNF8 expression levels alters the TRPC6 channel current and can modulate activation of NFAT-mediated transcription downstream of gain-of-function mutant TRPC6. Taken together, these results identify SNF8 as a novel regulator of TRPC6.
Transient receptor potential; Calcium channel; Protein-protein interaction; Calcineurin-NFAT signalingBackground
Transient receptor potential canonical channel 6 (TRPC6) is one of seven members of the canonical transient receptor potential (TRPC) family of calcium-permeable cation channels that allow for an increase in intracellular calcium following activation of G-protein coupled receptors and receptor tyrosine kinases [1]. TRPC channel subunits assemble to form homo- and/or hetero-tetramers [2]. Each TRPC subunit contains intracellular amino and carboxyl-terminal domains flanking six transmembrane domains; the intracellular sequences host canonical protein-protein interaction domains, including ankyrin-like repeats and a coiled-coil region in the amino-terminal cytoplasmic domain, and carboxyl-terminal TRP box, CIRB (calmodulin/IP3 receptor binding) sequence, and a coiled-coil region [3]. Multiple proteins have been shown to bind to TRPC6, including BKCa[4], calmodulin [5-7], FKBP12 [8], Fyn [9], IP3 receptor [7,10], MxA [11], the Na+/K+-ATPase pump [12], PLCγ [13], podocin [14,15], PTEN [16], and RNF24 [17]. In addition, TRPC6 channel activity can be modulated by tyrosine [9,13] and serine/threonine phosphorylation [18-21], and by binding of phosphoinositides [6,22,23]. However, how these various post-translational modifications and interacting partners ultimately alter channel activity has not been fully elucidated.
TRPC6 has been implicated in several physiological and pathophysiological functions in the kidney, heart and vasculature. Mutations in TRPC6 are a cause of familial, autosomal-dominant, adult-onset focal segmental glomerulosclerosis (FSGS), a form of proteinuric renal disease [15,24], and increased TRPC6 expression has been observed in acquired proteinuric kidney disease [25,26]. In contrast, TRPC6-deficient mice show mild protection against angiotensin II mediated glomerular damage [27]. The majority of FSGS-associated mutations appear to be gain-of-function, with increased channel current amplitudes and reduced current decay having been reported [15,24,28,29]. As all currently identified FSGS disease-associated TRPC6 mutations map to the intracellular domains of the channel, it has been hypothesized that these mutations may act by disrupting the binding of regulatory proteins to TRPC6, with the slit diaphragm protein nephrin proposed as one such protein [13]. How enhanced TRPC6 activity leads to renal disease, though, remains unknown. In contrast, in the heart upregulation of TRPC6 has been linked to the development of pathologic cardiac hypertrophy through activation of the calcineurin-NFAT pathway [30,31]. Finally, examinations of TRPC6-deficient mice have suggested roles in hypoxic pulmonary vasoconstriction [32], the development of pulmonary edema in response to ischemia-reperfusion injury [33], and platelet function [34].
To discover novel binding partners of TRPC6, we performed a yeast two-hybrid screen using the amino-terminal domain of TRPC6 as bait and isolated SNF8 as a TRPC6 binding partner. SNF8 is the mammalian counterpart to yeast VPS22, a component of the ESCRT-II endosomal trafficking complex required for the endocytosis and lysosomal degradation of transmembrane proteins [35-37]. The SNF8-TRPC6 interaction was mapped to the first 107 residues of the N-terminus of TRPC6, and was confirmed by co-immunoprecipitation as well as immunofluorescence microscopy. Additionally, overexpression and RNAi-mediated knockdown of SNF8 in tissue culture reveals a modulatory effect on wild-type and mutant TRPC6 channel activity as evidenced by whole-cell electrophysiology studies and transcriptional luciferase reporter assays.
SNF8 binds to the amino-terminus of TRPC6 in the Yeast Two-Hybrid System
The amino-terminal cytoplasmic domain of human TRPC6 (amino acids 1-406) was used as bait in a yeast two-hybrid screen to identify potential TRPC6 interacting proteins in a human kidney cDNA library. On the basis of both nutritional selection and β-galactosidase activity, 10 clones of SNF8 (also known as EAP30 and VPS22) were isolated; all clones included the entire coding sequence of SNF8 and varying lengths of 5′ UTR. Full-length SNF8 fused to the Gal4 activation domain was able to interact with the amino-terminal cytoplasmic domain, but not the carboxyl-terminal cytoplasmic domain of TRPC6 in the yeast two-hybrid system as assessed by growth on histidine and adenine free media (Figure 1) and β-galactosidase activity (data not shown). Using truncation mutants of the amino-terminal of TRPC6, it was found that amino acids 1-107, containing the sequence prior to the ankyrin repeats, are necessary and sufficient for this interaction (Figure 1).
Figure 1. SNF8 binding to TRPC6 in the two-hybrid assay. A. Yeast carrying the pGBKT7 plasmid encoding for the Gal4 DNA binding domain fused to the indicated TRPC6 amino acids, and either pGADT7 (encoding for the GAL4 activation domain alone) or pGADT7-SNF8 (expressing full-length SNF8 fused to the GAL4 activation domain) were grown on high stringency selective media, SD-ade, his, leu, trp (left panel), to assess for protein-protein interaction, or on SD-leu, trp (right panel) as a control. B. Schematic of TRPC6 structure indicating the location of various structural domains relative to the TRPC6 constructs used in the yeast-two hybrid assay.
TRPC6 and SNF8 interact in mammalian cells
To confirm that the interaction between TRPC6 and SNF8 can occur in a cellular context, cells stably expressing FLAG-tagged TRPC6 under a tetracycline inducible promoter were transfected with HA-tagged SNF8. TRPC6 was immunoprecipitated with the FLAG M2 monoclonal antibody, and the presence of SNF8 in the isolated immune complexes was assessed by Western blot using an anti-HA antibody (Figure 2A). HA-SNF8 was detected in the immunoprecipitated material only when co-expressed with FLAG-TRPC6. We have not been able to confirm the interaction by co-immunoprecipitation of endogenous SNF8 and TRPC6, but this may be due at least in part due to the limited availability of high quality antibodies to detect endogenous SNF8 (data now shown) or a relatively low affinity interaction between the two proteins.
Figure 2. Binding of TRPC6 to SNF8 in 293T cells. A. M1R cells expressing FLAG-tagged TRPC6 under a tetracycline inducible promoter were transfected with HA-tagged SNF8 or control vector, followed by culture in the absence or presence of 1 μg/ml tetracycline to induce the expression of TRPC6. Cell lysates were either directly analyzed for TRPC6 and SNF8 expression by Western blot (top and middle panel, respectively), or incubated with FLAG M2 affinity beads to immunoprecipitate TRPC6. HA-tagged SNF8 was detected bound to the affinity beads when expressed in the presence of TRPC6 (bottom panel). B. Cells transfected with HA-tagged SNF8 and induced to express TRPC6 as indicated were treated with or without 100 μM carbachol for 5 minutes prior to lysis and immunoprecipitation as in (A) above.
To assess if the interaction between the two proteins might be influenced by TRPC6 channel activation, cells co-expressing both proteins were treated with or without carbachol to activate TRPC6 prior to cell lysis and immunoprecipitation. No notable difference in the amount of SNF8 in the immune complexes was seen upon carbachol stimulation (Figure 2B). This suggests that channel activity or calcium influx is not required for the establishment of this interaction.
To further confirm the potential interaction between SNF8 and TRPC6, the localization of the two overexpressed proteins was determined by immunofluorescence microscopy. Overall, there was little colocalization of the two signals, though small punctate regions suggesting areas of colocalization of the two proteins were seen in most cells examined (Figure 3).
Figure 3. Immunofluorescence localization of TRPC6 and SNF8. M1R cells expressing FLAG-TRPC6 and HA-SNF8 were fixed and stained as follows: TRPC6 was detected with the anti-FLAG M2 mouse monoclonal antibody and an Alexa488 conjugated anti-mouse secondary (bottom left); SNF8 was detected with an anti-HA rabbit monoclonal antibody and a Cy3 conjugated anti-rabbit secondary (top right). Nuclei were counterstained with DAPI (top left). A merged image (bottom right) demonstrates partial localization of TRPC6 and SNF8 in punctuate structures (arrows). Bar = 10 μm.
SNF8 enhances TRPC6-mediated current
To address whether SNF8 may play a role in the regulation of TRPC6 activity, we performed electrophysiology experiments in the whole cell configuration in HEK293T cells expressing the muscarinic M1 acetylcholine receptor and either TRPC6 and SNF8, or TRPC6 alone. Activation of the M1 receptor with carbachol resulted in a whole cell current with a characteristic current-voltage relationship for TRPC6: a small inward and a steeply outwardly rectifying component. Co-expression of SNF8 led to increased whole cell current amplitude in both the inward and outward directions (Figure 4A and C). The ability of SNF8 to enhance TRPC6-mediated currents extended to channels containing an FSGS-associated mutation, E897K (Figure 4B and C). This mutation was previously characterized as a gain-of-function mutation leading to increased whole cell current amplitude compared to wild-type controls [15].
Figure 4. SNF8 enhances TRPC6-mediated currents. Whole-cell currents were measured in HEK cells expressing the M1 receptor and transiently transfected with either wild-type TRPC6 (A) or E897K mutant TRPC6 (B), alone (black line) or with SNF8 (red). Cells were stimulated with 100 μM carbachol to activate TRPC6-mediated current. C. Average normalized current density in response to carbachol. One way ANOVA Fisher LSD; ** p value <0.01 vs. TRPC6 WT, ## p value <0.01 between SNF8 and non-SNF8 expressing cells; n=6.
We attempted to ascertain whether the effect of SNF8 on TRPC6 was dependent on direct binding of the two proteins. A TRPC6 construct lacking the amino terminal 106 amino acids (FLAG-TRPC6 107-931), required for SNF8 binding in the two hybrid assay, expressed at significantly lower levels than full-length TRPC6, though it was capable of forming multimers, based on its ability to co-immunoprecipitate full-length HA-tagged TRPC6 (Figure 5A). However, in contrast to full length TRPC6, the truncated protein failed to generate current in response to OAG stimulation (Figure 5B-D), preventing us from addressing whether the effect of SNF8 on TRPC6 currents was dependent on binding at this site. Similarly, TRPC6 107-931 failed to generate significant current after stimulation with carbachol (data not shown).
Figure 5. Amino acids 1-106 are necessary for TRPC6 channel activity. A. Full-length HA-tagged TRPC6 was expressed together with the indicated FLAG-tagged TRPC6 constructs in HEK 293T cells. Expression of FLAG- and HA-tagged protein was confirmed by Western blot of whole cell lysates (top and middle panels). The ability of the amino-terminal truncated TRPC6 protein to multimerize with full-length TRPC6 was confirmed by FLAG immunoprecipitation followed by western-blotting for HA-TRPC6 (bottom panel). Whole cell currents in HEK cells transiently transfected with wild-type TRPC6 (B) or TRPC6 107-931 (C) under basal conditions and upon stimulation with 10 μM OAG. D. Average normalized current density in response to OAG. One way ANOVA ; * p value < 0.05; n=8 cells.
Modulating SNF8 levels alters NFAT-mediated transcription downstream of mutant TRPC6
We have previously demonstrated that several FSGS-associated mutations in TRPC6 lead to activation of calcineurin-NFAT signaling and NFAT-mediated transcription in the absence of exogenous stimuli [38]. In light of the ability of SNF8 to enhance TRPC6-mediated whole cell currents, we examined the ability of SNF8 overexpression to affect TRPC6 mediated activation of NFAT-mediated transcription (Figure 6A). Overexpression of SNF8 in cells expressing either the R895C or E897K mutant TRPC6 channels led to an increase in NFAT-mediated transcription under unstimulated conditions. In contrast, in cells expressing wild-type TRPC6, which does not activate NFAT-mediated transcription under unstimulated conditions, SNF8 overexpression did not lead to activation of NFAT-driven transcription. Unfortunately, known activators of TRPC6, including carbachol and OAG, are able to substantially increase NFAT-mediated transcription in 293T cells even in the absence of exogenously expressed TRPC6 [38]. Therefore, we have not been able to ascertain whether overexpression of SNF8 is able to enhance NFAT activation by wild-type TRPC6 after the channel has been activated. Taken together with the electrophysiologic data, these results suggest that SNF8 can enhance TRPC6-mediated currents and downstream signaling pathways, but is not sufficient to activate the channel.
Figure 6. SNF8 effect on NFAT-mediated transcription. A. Cells stably expressing tetracycline-inducible wild-type, R895C or E897K TRPC6 were transfected with an NFAT-responsive luciferase reporter plasmid and either control plasmid or HA-SNF8 expression plasmid. Normalized luciferase activity was determined after TRPC6 expression was induced for 24 hours. Results are means ± SEM; one way ANOVA; * p<0.05 vs. control; ***p<0.001 vs. control. B. Western blots to confirm the expression of FLAG-TRPC6 and HA-SNF8 in lysates from cells used in the luciferase assays in (A). Actin was utilized as a loading control. C. Cells expressing wild-type or R895C TRPC6 under a tetracycline inducible promoter were transfected with plasmid encoding for either SNF8 shRNA (SNF8 Knockdown) or a scrambled shRNA (control), and luciferase reporter plasmids. 24 hours after transfection, TRPC6 expression was induced by the addition of tetracycline as indicated (induced). Cells cultured in the absence of tetracycline (uninduced) did not express detectable amounts of TRPC6, and were used as controls. 24 hours post-induction, luciferase reporter assays were performed. Results are means ± SEM; one way ANOVA, ***p<0.001 vs control shRNA. D. M1R cells were transfected with plasmids encoding for either an shRNA targeting SNF8, or a scrambled shRNA (control), as well as GFP. 48 hours post-transfection, cells were trypsinized and GFP expressing cells isolated by FACS cell sorting. Lysates from GFP positive cells were analyzed by Western blot for expression of SNF8. Actin expression was used as a loading control.
Most of our current knowledge regarding SNF8 relates to its function as part of the ESCRT-II complex involved in transmembrane protein trafficking into multivesiculated bodies [37,39,40]. We therefore hypothesized that overexpression of SNF8, rather than increasing ESCRT-II activity, might act in a dominant negative fashion by providing monomeric SNF8 to compete with intact ESCRT-II complex for TRPC6 binding. To address this possibility, we made use of a short hairpin RNA expressing plasmid to knock down SNF8 protein levels in M1R cells. Introducing the SNF8 shRNA expressing plasmid into cells expressing R895C mutant TRPC6 led to a modest decrease in activity from an NFAT-responsive luciferase reporter construct, compared to cells transfected with a control shRNA plasmid (Figure 6C). Expression of the SNF8 shRNA did not significantly alter basal NFAT-mediated transcription in control cells (WT uninduced and R895C uninduced) or expressing wild-type TRPC6 (WT induced). Western blot analysis confirmed a decrease in endogenous SNF8 levels with the SNF8 shRNA expression plasmid compared to control (Figure 6). These results suggest that: 1. the effects of overexpressed SNF8 on TRPC6 channel activity and signaling are not mediated by a dominant negative effect, and 2. while SNF8 may enhance TRPC6 activity, it is likely not required for channel activity given the only modest decrease in NFAT-reporter activity in response to SNF8 knockdown in R895C expressing cells. Confirmation of this later point will require the development of cells completely devoid of SNF8.
SNF8 overexpression does not alter TRPC6 content on the cell surface or in detergent resistant membranes
Several TRP channels, including TRPC6, have been reported to be regulated by altering their cell surface expression through regulated endo- and exocytosis [13,41,42]. Mutations in snf8 have been reported to affect the transport kinetics of Ena1p, a sodium pump in yeast [43]. We therefore wished to examine whether SNF8 overexpression might alter TRPC6 function by modulating the amount of channel expressed on the plasma membrane. M1R cells were transiently transfected with expression plasmids for FLAG-TRPC6 and HA-SNF8, either in combination, or alone with control plasmid. After surface biotinylation of the cells, the amount of TRPC6 bound to streptavidin beads (representing cell surface associated protein) was compared to total TRPC6 in whole cell lysates (Figure 7). Co-expression of SNF8 did not appreciably alter the amount of TRPC6 bound to the streptavidin beads compared to total TRPC6 levels. Interestingly, small amounts of SNF8 could also be detected in material bound to streptavidin beads (lower panels), consistent with its recruitment to transmembrane proteins, including plasma membrane proteins. However, the amount of SNF8 pulled down by streptavidin beads was not appreciably altered by the presence or absence of TRPC6, suggesting that cell surface proteins other than TRPC6 were largely responsible for this effect.
Figure 7. Surface expression of TRPC6. HEK293T cells were transiently transfected with the indicated combinations of FLAG-TRPC6 (FLAG-C6) and HA-SNF8 expression plasmids. 48 hours after transfection, cells were surface biotinylated or mock biotinylated (-) as indicated and lysed. Biotinylated protein and associated proteins were isolated by binding to streptavidin agarose beads. The amounts of TRPC6 and SNF8 in whole lysate (left panel) and bound to streptavidin beads (right panel) were assessed by Western blotting.
Several TRPC proteins have been reported to partition into detergent-resistant membranes [44-50], and TPRC1 binds to caveolin-1, a lipid raft-associated protein [45,47-49,51-53]. Furthermore, STIM1 overexpression was reported to shift TRPC1 into lipid rafts, thereby altering its channel properties [52,54,55]. We therefore examined whether SNF8 may alter the amount of TRPC6 present in detergent-resistant membranes. In control cells, only a very small fraction of TRPC6 (Figure 8, top panel) was detected in the fractions containing detergent-resistant membranes, as assayed by the presence of the lipid raft marker caveolin-1 (Figure 8A, bottom panel), similar to previously reported results [46,50]. The amount of total TRPC6 found in the low density fractions did not change appreciably when HA-SNF8 was overexpressed (Figure 8B). A small amount of SNF8 was also detected in the detergent resistant membranes (Figure 8B, middle panel). It remains unclear why caveolin-1 localization peaked in fraction 2, while TRPC6 and SNF8 in the low density fractions were mainly in fraction 3, but the generation of distinct lipid raft subfractions has been reported using a protocol similar to the one used here [56].
Figure 8. Detergent-resistant membrane preparation. M1R cells stably expressing TRPC6 were transiently transfected with control vector (A) or HA-SNF8 expression construct (B). Triton X-100 detergent resistant membranes were separated from other proteins by sucrose gradient centrifugation, and the abundance of FLAG-TRPC6 and HA-SNF8 in each fraction analyzed by Western blot. Caveolin-1 represents a marker of lipid rafts. Fraction 1 represents the top of the gradient.
Here we identify SNF8 as a novel binding partner of TRPC6. The interaction was first established in a yeast two-hybrid screen, where it was found to be mediated by the amino terminal 107 amino acids of TRPC6. The TRPC6-SNF8 interaction has been confirmed by co-immunoprecipitation, and the proteins partially colocalize in cells. Importantly, SNF8 overexpression enhances whole cell current amplitudes mediated by both wild-type and FSGS-associated mutant TRPC6, as well as mutant TRPC6-mediated NFAT activation, while RNAi mediated knock-down of endogenous SNF8 diminishes NFAT activation by mutant TRPC6. The effect of SNF8 on TRPC6 channel activity and downstream activation of NFAT-mediated transcription does not appear to be mediated by alterations in the global surface expression levels of TRPC6 or a shift of channel into lipid rafts. Taken together, these results suggest a novel role for SNF8 in the modulation of TRPC6 channel activity.
It is difficult to reconcile the ability of SNF8 to enhance TRPC6-mediated current with its major known function, sorting transmembrane proteins into multivesiculated bodies for degradation. SNF8, together with VPS25 and VPS36, forms the endosomal sorting complex for transport (ESCRT) -II complex [35]. ESCRT-II, in conjunction with the ESCRT-I and –III complexes, is involved in the budding of intraluminal vesicles from endosomes to form multivesiculated bodies (MVB) (reviewed in [37,39,40]). This process is critical for the downregulation of multiple signaling receptors, including the yeast mating receptor Ste2 [35], EGFR[36,57] and Notch [58,59]. The ESCRT complexes not only target ubiquitinated transmembrane proteins to the vesicles of the MVB, but induce the necessary membrane curvature and ultimate scission of the vesicles from their parent membrane [60-62]. In addition to their role in MVB formation, ESCRTs are involved in the membrane scission required for cytokinesis and the budding of enveloped viruses [63,64].
Whether the endosomal-lysosomal functions of SNF8 might relate to TRPC6 activity is unclear. It is worth noting that SNF8 has been implicated to have several non-endosomal functions. These include the establishment of the bicoid mRNA gradient in the Drosophila oocyte [65] and modulation of the ELL transcription elongation complex [66]. Perhaps most relevant to its role in enhancing TRPC6-mediated currents, the yeast homologue of SNF8, VPS22, as well as other members of the ESCRT complexes, have been shown to be involved in the trafficking and surface expression of the sodium pump Ena1 [43]. Although we have not been able to detect a change in the amount of total TRPC6 expressed on the cell surface or in lipid rafts in response to co-expression of SNF8, it is possible that SNF8 traffics the channel to a subdomain of the plasma membrane where TRPC6 activity is enhanced. Along these lines, it is noteworthy that podocin enhances TRPC6 activity in a cholesterol dependent manner without altering plasma membrane expression [14], while the differential requirement of ESCRT-II for the budding of avian sarcoma and leukosis virus (ASLV) and human immunodeficiency virus, type-1 (HIV-1), correlates with their assembly on phosphatidylethanolamine (PE) containing or PE-negative membranes, respectively [63,67]. Alternatively, it is possible that SNF8 regulates TRPC6 indirectly by competing with a negative regulator or by altering the activity of another membrane protein in the vicinity of TRPC6, such as an enzyme that affects phosphoinositide levels. Finally, TRPC6 has been reported to be activated by membrane deformation [68], though this has been disputed [69]. One could hypothesize that SNF8 may act to recruit ESCRT-II to TRPC6, and alter TRPC6 function through local convex deformation of the membrane [70]. Further understanding the mechanism whereby SNF8 enhances TRPC6 currents will be the goal of future investigations.
This work shows that: 1. SNF8 is a potential binding partner of TRPC6, 2. overexpression of SNF8 enhances both wild-type and mutant TRPC6 current densities, and 3. modulating SNF8 expression levels affects NFAT activation downstream of gain-of-function, FSGS-associated TRPC6 mutations. The mechanism for regulating channel activity is not mediated by changes in global cell surface expression or recruitment into lipid rafts. Taken together, these results identify SNF8 as a potent modulator of the TRPC6 channel.
Plasmids and reagents
The human TRPC6 coding sequence, with or without mutations as outlined in the text, and containing an amino-terminal FLAG tag sequence, was cloned into pcDNA4/TO/myc-HIS B (Clontech) using standard PCR-based techniques. Similarly, full-length human TRPC6 carrying an amino-terminal HA tag was amplified by PCR and subcloned into pcDNA3.1. The HA-SNF8 expression plasmid was a gift from C. Bucci [71]. The Matchmaker Two-Hybrid System and S. cerevisiae Y187 pre-transformed with a human kidney cDNA library were purchased from Clontech (Palo Alto, CA). The dual luciferase assay kit and reporter vectors pGL4.30 and pGL4.74 were obtained from Promega. Affinity purified rabbit anti-TRPC6 polyclonal antibody was purchased from Chemicon, anti-FLAG M2 monoclonal antibody and anti-FLAG rabbit polyclonal antibodies were purchased from Sigma, rabbit anti-GFP polyclonal antibody and mouse anti-HA monoclonal antibody were purchased from Abcam Inc, and rabbit anti-HA monoclonal antibody (C29F4) was purchased from Cell Signaling Technologies. Anti-SNF8 rabbit polyclonal antibody was the kind gift of Dr. H. Stenmark [36]. Anti-caveolin-1 mouse monoclonal antibody (clone 2297) was obtained from BD Biosciences.
Yeast two-hybrid screen
cDNA encoding residues 1 through 406 of TRPC6 (wild-type N-terminal domain) was used as bait and cloned in-frame with GAL4 DNA-binding domain in the vector pGBKT7-BD and transformed into yeast strain AH109. The bait strain was mated to Y187 yeast strain pre-transformed with a commercially available human kidney cDNA library cloned into pACT2-AD vector according to the manufacturer's protocol (Clontech). Based on mating efficiency, 1 × 106 clones were screened. Mated yeast cells were grown on high-stringency selection plates (SD-Leu, Trp, Ade, His) and positive colonies were further verified by growth on high-stringency plates supplemented with X-α-gal as a test for β-galactosidase activity. pACT2-AD plasmids containing library inserts from positive colonies were isolated and transformed into DH5α derived E. coli (New England Biolabs). Plasmids were then isolated from bacteria, sequenced, and analyzed using the Blast-NT alignment algorithm from NCBI.
To confirm the interaction, the full-length SNF8 coding sequence was cloned inframe into the pGAD-T7 plasmid and the resulting plasmid transformed into Y187 strain yeast. The resulting strain was mated with AH109 bait strain harboring pGBKT7 with wild-type TRPC6 N-terminal sequences or the TRPC6 C-terminal domain (corresponding to amino acids 726-931). The resulting diploids were tested for positive interaction by growth on high-stringency plates.
Cell culture and luciferase assays
Cells stably expressing the M1 muscarinic acetylcholine receptor and either wild-type, R895C or E897K TRPC6 under a tetracycline-inducible promoter were generated from T-Rex-293 cells (Invitrogen), as previously described [38]. Stable cell lines were induced to express TRPC6 for 24 hours prior to lysis by adding tetracycline to a 1 μg/ml final concentration in culture medium. Luciferase assays were carried out essentially as previously described [38] using the dual luciferase reporter assay (DLR; Roche) using a Veritas microplate luminometer (Turner Biosystems). All conditions were tested in triplicate and results normalized to the Renilla luciferase internal control.
RNA interference
SNF8 was knocked down using short hairpin-expressing double-stranded oligonucleotides cloned into pcDNA 6.2-GW/EmGFP-miR plasmids (BLOCK-iT™ Pol II miR RNAi Expression Vector Kit, Invitrogen). A specific shRNA sequence against SNF8 (sense 5′-TGACTTCG[CC]CAATGTCAGTCAA-3′ and antisense 5′-TTGACTGACATCCTGGGCGAA-3′), and a scrabbled control sequence, were obtained from Invitrogen and cloned into the plasmid. Knock-down efficiency was determined by transient transfection of M1R cells with either SNF8 or control plasmid, followed by FACS sorting for GFP expression to isolate the transfected cells after 48 hrs. SNF8 expression in sorted cells was assessed by immunbloting for SNF8.
To assess the effect of SNF8 knock-down on NFAT-mediated transcription, cells were transfected with the indicated shRNA expression vector and luciferase reporter plasmids using Fugene 6. 24 hours after transfection, TRPC6 expression was induced where indicated by the addition of tetracycline (1μg/ml final concentration) to the media. After an additional 24 hours, cells were lysed and processed for dual luciferase reporter assay.
Immunoprecipitation and Immunoblotting
Transfected cells were rinsed once in phosphate buffered saline and lysed in modified RIPA lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate supplemented with Complete protease inhibitor cocktail (Roche)). After clearing lysates by centrifugation at 14,000 RPM for 15 minutes at 4ºC, supernatants were incubated with 30 μl of FLAG M2 agarose slurry (Sigma-Aldrich), and incubated with constant agitation at 4ºC for 2-3 hours. Immunoprecipitated complexes were washed three times with lysis buffer and eluted off of the beads by boiling in SDS-sample loading buffer.
Cell lysates and immunoprecipitated materials were separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk in PBST (PBS with 0.05% Tween-20) for 1 hour at room temperature, followed by overnight incubation in 1:1000 anti-TRPC6, 1:500 anti-HA, 1:500 FLAG M2, or 1:200 anti-SNF8 antibody in 5% non-fat milk PBST. After three washes in PBST, blots were incubated with the appropriate secondary antibody conjugated to HRP (Pierce) in PBST at room temperature, followed by detection with SuperSignal West Pico chemiluminescent substrate (Pierce).
Fluorescence microscopy
Cells stably expressing wild-type TRPC6 under a tetracycline inducible promoter were grown on collagen I coated glass coverslips and transfected with plasmid encoding for HA-SNF8 using Fugene6 followed by treatment with tetracycline to induce FLAG-TRPC6. 24 hours after inducing TRPC6 expression, cells were washed with PBS, fixed in 2% paraformaldehyde, 4% sucrose in PBS, and permeabilized with 0.3% Triton X-100 in PBS. Nonspecific binding sites were blocked using blocking solution (2% bovine serum albumin, 2% fetal calf serum, 0.2% fish gelatin). Cells were incubated with 1:200 anti-FLAG M2 antibody and 1:1600 anti-HA rabbit monoclonal antibody, followed by Alexa488 conjugated goat anti-mouse and Cy3 conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch), and mounted on slides with ProLong Gold antifade reagent (Invitrogen). Images were taken using a Zeiss LSM 510 confocal microscope.
Patch-clamp electrophysiology (Axopatch 200B amplifier, Axon Instruments, CA) was performed in the whole-cell configuration. Briefly, HEK293T cells (American Type Culture Collection, VA) were plated on glass coverslips at low density and placed in the recording chamber. The patch pipettes with resistances of 3–4 MΩ were pulled from borosilicate glass with a P-97 puller (Sutter Instrument) and filled with a solution containing (in mM): 135 CH3SO3Cs, 10 CsCl, 3 MgATP, 0.2 NaGTP, 0.2 EGTA, 0.13 CaCl2, and 10 HEPES, pH 7.3 with CsOH. The bath solution contained (in mM): 135 CH3SO3Na, 5 CsCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 Glucose, pH 7.4 with NaOH. Carbachol (100 μM) or 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10 μM) was applied to the bath solution. Whole-cell currents were recorded from –100 mV to 100 mV voltage ramps over 150 ms and a holding potential of 0 mV. All data were acquired at room temperature and analyzed using pClamp 10 (Axon Instruments, CA). Statistical analysis was done using one-way ANOVA Fisher's LSD test, and p values <0.05 were considered significant.
Surface biotinylation
Cells were surface biotinylated on ice using Sulfo-NHS-SS-biotin (Pierce) 48 hours after transfection, essentially as previously described [38], followed by lysis in modified RIPA buffer. Lysates were incubated with streptavidin beads (Pierce) at 4°C. The streptavidin beads were extensively washed and bound material was eluted and analyzed by SDS-PAGE electrophoresis followed by Western blot analysis.
Lipid raft preparation
M1R cells stably expressing FLAG-tagged TRPC6 were transfected with control or HA-SNF8 expression constructs. 48 hours after transfection, cells were processed using a protocol modified from Alicia et al [54]. Briefly, cells were washed in ice cold PBS, scraped off of plates in PBS and lysed in 1% Triton X-100 in MES buffer (25mM MES, pH 6.5, 150mM NaCl, 2mM EDTA complemented with protease inhibitors) on ice for 20 minutes. Lysates were further homogenized in a glass homogenizer with the loose fitting piston, then mixed with an equal volume of 85% (w/v) sucrose in MES buffer. Lysates were overlayed with 4mls of 40% sucrose in MES buffer followed by 4mls of 5% sucrose in MES buffer. Lysates were spun at 175,000x g for 24 hours in an SW41 Ti rotor. Eight 1.5 ml fractions were obtained by aspiration starting at the top of the gradient. Aliqouts of each fraction were combined with 4x sample loading buffer with β-mercaptoethanol and analyzed by Western blot as above.
ASLV: Avian sarcoma and leukosis virus; ESCRT: Endosomal sorting complex for transport; FSGS: Focal segmental glomerulosclerosis; GFP: Green fluorescent protein; HA: Hemaglutinin; HIV: Human immunodeficiency virus; NFAT: Nuclear factor of activated T-cells; PAGE: Poly-accrylamide gel electrophoresis, PE, Phosphatidylethanolamine; shRNA: Short hairpin ribonucleic acid; TRPC: Canonical transient receptor potential.
Competing interests
The authors declare that they have no competing interest.
Authors' contributions
RC performed the yeast-two hybrid screen and candidate identification, generated shRNA plasmids, performed SNF8 knockdown experiments and luciferase assays, and helped draft the manuscript. SK generated expression constructs, and performed cell transfections and immunoblotting. MRP participated in the design and coordination of the study. DT and AG performed all electrophysiology experiments and related statistical analysis, and helped draft the manuscript. JS conceived of the study, performed co-immunoprecipitation, immunofluorescence, surface biotinylation, membrane fractionation and luciferase assays, performed statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by NIH grants DK743082 and DK080947 and an ASN Young Investigator Award (to J.S.), by NIH grant DK59588 (to M.R.P.), a Graduate Prize Fellowship from the Harvard University Graduate School of Arts and Sciences and a grant from the Office of Enrichment Programs at Harvard Medical School (to R.C.), and by a Nephcure Young Investigator Award, an ASN Young Investigator Award and NIH grant DK083511 (to A.G.).
We are grateful to Dr. Cecilia Bucci for the HA-SNF8 expression construct and to Dr. Harald Stenmark for the kind gift of anti-SNF8 antibody. We would like to thank Arnolt Ramos for assistance with electrophysiology experiments. We appreciate the assistance of the Beth Israel Deaconess Confocal Imaging Core and the Dana Farber Cancer Center Flow Cytometry Core facilities. We thank the members of the Pollak lab for helpful discussions and suggestions.
Montell C: The TRP superfamily of cation channels.
Sci STKE 2005, 2005(272):re3. PubMed Abstract | Publisher Full Text
Hofmann T, Schaefer M, Schultz G, Gudermann T: Subunit composition of mammalian transient receptor potential channels in living cells.
Proc Natl Acad Sci U S A 2002, 99(11):7461-7466. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Vazquez G, Wedel BJ, Aziz O, Trebak M, Putney JW Jr: The mammalian TRPC cation channels.
Biochim Biophys Acta 2004, 1742(1–3):21-36. PubMed Abstract | Publisher Full Text
Kim EY, Alvarez-Baron CP, Dryer SE: Canonical transient receptor potential channel (TRPC)3 and TRPC6 associate with large-conductance Ca2+-activated K+ (BKCa) channels: role in BKCa trafficking to the surface of cultured podocytes.
Mol Pharmacol 2009, 75(3):466-477. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Boulay G: Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells.
Cell Calcium 2002, 32(4):201-207. PubMed Abstract | Publisher Full Text
Kwon Y, Hofmann T, Montell C: Integration of phosphoinositide- and calmodulin-mediated regulation of TRPC6.
Mol Cell 2007, 25(4):491-503. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tang J, Lin Y, Zhang Z, Tikunova S, Birnbaumer L, Zhu MX: Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels.
J Biol Chem 2001, 276(24):21303-21310. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sinkins WG, Goel M, Estacion M, Schilling WP: Association of immunophilins with mammalian TRPC channels.
J Biol Chem 2004, 279(33):34521-34529. PubMed Abstract | Publisher Full Text
Hisatsune C, Kuroda Y, Nakamura K, Inoue T, Nakamura T, Michikawa T, Mizutani A, Mikoshiba K: Regulation of TRPC6 channel activity by tyrosine phosphorylation.
J Biol Chem 2004, 279(18):18887-18894. PubMed Abstract | Publisher Full Text
Boulay G, Brown DM, Qin N, Jiang M, Dietrich A, Zhu MX, Chen Z, Birnbaumer M, Mikoshiba K, Birnbaumer L: Modulation of Ca(2+) entry by polypeptides of the inositol 1,4, 5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca(2+) entry.
Proc Natl Acad Sci U S A 1999, 96(26):14955-14960. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lussier MP, Cayouette S, Lepage PK, Bernier CL, Francoeur N, St-Hilaire M, Pinard M, Boulay G: MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC.
J Biol Chem 2005, 280(19):19393-19400. PubMed Abstract | Publisher Full Text
Goel M, Sinkins W, Keightley A, Kinter M, Schilling WP: Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na(+)/K(+)-ATPase.
Pflugers Arch 2005, 451(1):87-98. PubMed Abstract | Publisher Full Text
Kanda S, Harita Y, Shibagaki Y, Sekine T, Igarashi T, Inoue T, Hattori S: Tyrosine phosphorylation-dependent activation of TRPC6 regulated by PLC-gamma1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis.
Mol Biol Cell 2011, 22(11):1824-1835. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Huber TB, Schermer B, Muller RU, Hohne M, Bartram M, Calixto A, Hagmann H, Reinhardt C, Koos F, Kunzelmann K, et al.: Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels.
Proc Natl Acad Sci U S A 2006, 103(46):17079-17086. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Reiser J, Polu KR, Moller CC, Kenlan P, Altintas MM, Wei C, Faul C, Herbert S, Villegas I, Avila-Casado C, et al.: TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function.
Nat Genet 2005, 37(7):739-744. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kini V, Chavez A, Mehta D: A new role for PTEN in regulating transient receptor potential canonical channel 6-mediated Ca2+ entry, endothelial permeability, and angiogenesis.
J Biol Chem 2010, 285(43):33082-33091. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lussier MP, Lepage PK, Bousquet SM, Boulay G: RNF24, a new TRPC interacting protein, causes the intracellular retention of TRPC.
Cell Calcium 2008, 43(5):432-443. PubMed Abstract | Publisher Full Text
Shi J, Mori E, Mori Y, Mori M, Li J, Ito Y, Inoue R: Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells.
J Physiol 2004, 561(Pt 2):415-432. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kim JY, Saffen D: Activation of M1 muscarinic acetylcholine receptors stimulates the formation of a multiprotein complex centered on TRPC6 channels.
J Biol Chem 2005, 280(36):32035-32047. PubMed Abstract | Publisher Full Text
Takahashi S, Lin H, Geshi N, Mori Y, Kawarabayashi Y, Takami N, Mori MX, Honda A, Inoue R: Nitric oxide-cGMP-protein kinase G pathway negatively regulates vascular transient receptor potential channel TRPC6.
J Physiol 2008, 586(Pt 17):4209-4223. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bousquet SM, Monet M, Boulay G: Protein kinase C-dependent phosphorylation of transient receptor potential canonical 6 (TRPC6) on serine 448 causes channel inhibition.
J Biol Chem 2010, 285(52):40534-40543. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lemonnier L, Trebak M, Putney JW Jr: Complex regulation of the TRPC3, 6 and 7 channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate.
Cell Calcium 2008, 43(5):506-514. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Monet M, Francoeur N, Boulay G: Involvement of Phosphoinositide 3-Kinase and PTEN Protein in Mechanism of Activation of TRPC6 Protein in Vascular Smooth Muscle Cells.
J Biol Chem 2012, 287(21):17672-17681. PubMed Abstract | Publisher Full Text
Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, Daskalakis N, Kwan SY, Ebersviller S, Burchette JL, et al.: A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis.
Science 2005, 308(5729):1801-1804. PubMed Abstract | Publisher Full Text
Moller CC, Wei C, Altintas MM, Li J, Greka A, Ohse T, Pippin JW, Rastaldi MP, Wawersik S, Schiavi S, et al.: Induction of TRPC6 channel in acquired forms of proteinuric kidney disease.
J Am Soc Nephrol 2007, 18(1):29-36. PubMed Abstract | Publisher Full Text
Wang Z, Wei X, Zhang Y, Ma X, Li B, Zhang S, Du P, Zhang X, Yi F: NADPH oxidase-derived ROS contributes to upregulation of TRPC6 expression in puromycin aminonucleoside-induced podocyte injury.
Cell Physiol Biochem 2009, 24(5–6):619-626. PubMed Abstract | Publisher Full Text
Eckel J, Lavin PJ, Finch EA, Mukerji N, Burch J, Gbadegesin R, Wu G, Bowling B, Byrd A, Hall G, et al.: TRPC6 enhances angiotensin II-induced albuminuria.
J Am Soc Nephrol 2011, 22(3):526-535. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Heeringa SF, Moller CC, Du J, Yue L, Hinkes B, Chernin G, Vlangos CN, Hoyer PF, Reiser J, Hildebrandt F: A novel TRPC6 mutation that causes childhood FSGS.
PLoS One 2009, 4(11):e7771. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Zhu B, Chen N, Wang ZH, Pan XX, Ren H, Zhang W, Wang WM: Identification and functional analysis of a novel TRPC6 mutation associated with late onset familial focal segmental glomerulosclerosis in Chinese patients.
Mutat Res 2009, 664(1–2):84-90. PubMed Abstract | Publisher Full Text
Onohara N, Nishida M, Inoue R, Kobayashi H, Sumimoto H, Sato Y, Mori Y, Nagao T, Kurose H: TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy.
EMBO J 2006, 25(22):5305-5316. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kuwahara K, Wang Y, McAnally J, Richardson JA, Bassel-Duby R, Hill JA, Olson EN: TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling.
J Clin Invest 2006, 116(12):3114-3126. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, Olschewski A, Storch U, Mederos y Schnitzler M, Ghofrani HA, et al.: Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange.
Proc Natl Acad Sci U S A 2006, 103(50):19093-19098. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Weissmann N, Sydykov A, Kalwa H, Storch U, Fuchs B, Mederos y Schnitzler M, Brandes RP, Grimminger F, Meissner M, Freichel M, et al.: Activation of TRPC6 channels is essential for lung ischaemia-reperfusion induced oedema in mice.
Nat Commun 2012, 3:649. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Paez Espinosa EV, Murad JP, Ting HJ, Khasawneh FT: Mouse transient receptor potential channel 6: role in hemostasis and thrombogenesis.
Biochem Biophys Res Commun 2012, 417(2):853-856. PubMed Abstract | Publisher Full Text
Babst M, Katzmann DJ, Snyder WB, Wendland B, Emr SD: Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body.
Dev Cell 2002, 3(2):283-289. PubMed Abstract | Publisher Full Text
Malerod L, Stuffers S, Brech A, Stenmark H: Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation.
Traffic 2007, 8(11):1617-1629. PubMed Abstract | Publisher Full Text
Raiborg C, Stenmark H: The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins.
Nature 2009, 458(7237):445-452. PubMed Abstract | Publisher Full Text
Schlondorff J, Del Camino D, Carrasquillo R, Lacey V, Pollak MR: TRPC6 mutations associated with focal segmental glomerulosclerosis cause constitutive activation of NFAT-dependent transcription.
Am J Physiol Cell Physiol 2009, 296(3):C558-C569. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hanson PI, Shim S, Merrill SA: Cell biology of the ESCRT machinery.
Curr Opin Cell Biol 2009, 21(4):568-574. PubMed Abstract | Publisher Full Text
Wollert T, Yang D, Ren X, Lee HH, Im YJ, Hurley JH: The ESCRT machinery at a glance.
J Cell Sci 2009, 122(Pt 13):2163-2166. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bezzerides VJ, Ramsey IS, Kotecha S, Greka A, Clapham DE: Rapid vesicular translocation and insertion of TRP channels.
Nat Cell Biol 2004, 6(8):709-720. PubMed Abstract | Publisher Full Text
Cayouette S, Lussier MP, Mathieu EL, Bousquet SM, Boulay G: Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq protein-coupled receptor activation.
J Biol Chem 2004, 279(8):7241-7246. PubMed Abstract | Publisher Full Text
Logg K, Warringer J, Hashemi SH, Kall M, Blomberg A: The sodium pump Ena1p provides mechanistic insight into the salt sensitivity of vacuolar protein sorting mutants.
Biochim Biophys Acta 2008, 1783(6):974-984. PubMed Abstract | Publisher Full Text
Aires V, Hichami A, Boulay G, Khan NA: Activation of TRPC6 calcium channels by diacylglycerol (DAG)-containing arachidonic acid: a comparative study with DAG-containing docosahexaenoic acid.
Biochimie 2007, 89(8):926-937. PubMed Abstract | Publisher Full Text
Brazer SC, Singh BB, Liu X, Swaim W, Ambudkar IS: Caveolin-1 contributes to assembly of store-operated Ca2+ influx channels by regulating plasma membrane localization of TRPC1.
J Biol Chem 2003, 278(29):27208-27215. PubMed Abstract | Publisher Full Text
Brownlow SL, Sage SO: Transient receptor potential protein subunit assembly and membrane distribution in human platelets.
Thromb Haemost 2005, 94(4):839-845. PubMed Abstract | Publisher Full Text
Kwiatek AM, Minshall RD, Cool DR, Skidgel RA, Malik AB, Tiruppathi C: Caveolin-1 regulates store-operated Ca2+ influx by binding of its scaffolding domain to transient receptor potential channel-1 in endothelial cells.
Mol Pharmacol 2006, 70(4):1174-1183. PubMed Abstract | Publisher Full Text
Lockwich TP, Liu X, Singh BB, Jadlowiec J, Weiland S, Ambudkar IS: Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains.
J Biol Chem 2000, 275(16):11934-11942. PubMed Abstract | Publisher Full Text
Murata T, Lin MI, Stan RV, Bauer PM, Yu J, Sessa WC: Genetic evidence supporting caveolae microdomain regulation of calcium entry in endothelial cells.
J Biol Chem 2007, 282(22):16631-16643. PubMed Abstract | Publisher Full Text
Pani B, Singh BB: Lipid rafts/caveolae as microdomains of calcium signaling.
Cell Calcium 2009, 45(6):625-633. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Jardin I, Salido GM, Rosado JA: Role of lipid rafts in the interaction between hTRPC1, Orai1 and STIM1.
Channels (Austin) 2008, 2(6):401-403. Publisher Full Text
Pani B, Ong HL, Liu X, Rauser K, Ambudkar IS, Singh BB: Lipid rafts determine clustering of STIM1 in endoplasmic reticulum-plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE).
J Biol Chem 2008, 283(25):17333-17340. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sundivakkam PC, Kwiatek AM, Sharma TT, Minshall RD, Malik AB, Tiruppathi C: Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells.
Am J Physiol Cell Physiol 2009, 296(3):C403-C413. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Alicia S, Angelica Z, Carlos S, Alfonso S, Vaca L: STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts.
Cell Calcium 2008, 44(5):479-491. PubMed Abstract | Publisher Full Text
Pani B, Ong HL, Brazer SC, Liu X, Rauser K, Singh BB, Ambudkar IS: Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1.
Proc Natl Acad Sci U S A 2009, 106(47):20087-20092. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mellgren RL: Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.
J Biochem Biophys Methods 2008, 70(6):1029-1036. PubMed Abstract | Publisher Full Text
Langelier C, von Schwedler UK, Fisher RD, De Domenico I, White PL, Hill CP, Kaplan J, Ward D, Sundquist WI: Human ESCRT-II complex and its role in human immunodeficiency virus type 1 release.
J Virol 2006, 80(19):9465-9480. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Herz HM, Woodfield SE, Chen Z, Bolduc C, Bergmann A: Common and distinct genetic properties of ESCRT-II components in Drosophila.
PLoS One 2009, 4(1):e4165. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Vaccari T, Rusten TE, Menut L, Nezis IP, Brech A, Stenmark H, Bilder D: Comparative analysis of ESCRT-I, ESCRT-II and ESCRT-III function in Drosophila by efficient isolation of ESCRT mutants.
J Cell Sci 2009, 122(Pt 14):2413-2423. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hanson PI, Roth R, Lin Y, Heuser JE: Plasma membrane deformation by circular arrays of ESCRT-III protein filaments.
J Cell Biol 2008, 180(2):389-402. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Im YJ, Wollert T, Boura E, Hurley JH: Structure and function of the ESCRT-II-III interface in multivesicular body biogenesis.
Dev Cell 2009, 17(2):234-243. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Saksena S, Wahlman J, Teis D, Johnson AE, Emr SD: Functional reconstitution of ESCRT-III assembly and disassembly.
Cell 2009, 136(1):97-109. PubMed Abstract | Publisher Full Text
Pincetic A, Leis J: The Mechanism of Budding of Retroviruses From Cell Membranes.
Adv Virol 2009, 2009:6239691-6239699. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
McDonald B, Martin-Serrano J: No strings attached: the ESCRT machinery in viral budding and cytokinesis.
J Cell Sci 2009, 122(Pt 13):2167-2177. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Irion U, St Johnston D: Bicoid RNA localization requires specific binding of an endosomal sorting complex.
Nature 2007, 445(7127):554-558. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Schmidt AE, Miller T, Schmidt SL, Shiekhattar R, Shilatifard A: Cloning and characterization of the EAP30 subunit of the ELL complex that confers derepression of transcription by RNA polymerase II.
J Biol Chem 1999, 274(31):21981-21985. PubMed Abstract | Publisher Full Text
Medina G, Pincetic A, Ehrlich LS, Zhang Y, Tang Y, Leis J, Carter CA: Tsg101 can replace Nedd4 function in ASV Gag release but not membrane targeting.
Virology 2008, 377(1):30-38. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Spassova MA, Hewavitharana T, Xu W, Soboloff J, Gill DL: A common mechanism underlies stretch activation and receptor activation of TRPC6 channels.
Proc Natl Acad Sci U S A 2006, 103(44):16586-16591. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gottlieb P, Folgering J, Maroto R, Raso A, Wood TG, Kurosky A, Bowman C, Bichet D, Patel A, Sachs F, et al.: Revisiting TRPC1 and TRPC6 mechanosensitivity.
Pflugers Arch 2008, 455(6):1097-1103. PubMed Abstract | Publisher Full Text
Wollert T, Hurley JH: Molecular mechanism of multivesicular body biogenesis by ESCRT complexes.
Nature 2010, 464(7290):864-869. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Progida C, Spinosa MR, De Luca A, Bucci C: RILP interacts with the VPS22 component of the ESCRT-II complex.
Biochem Biophys Res Commun 2006, 347(4):1074-1079. PubMed Abstract | Publisher Full Text


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[ Music Index | Metro Santa Cruz | MetroActive Central | Archives ]
Life Is a CabMuFest
Paul Schraub
The annual festival ushers in its 36th season with a return to the sonorous basics
By Philip Collins
AUGUST IS HERE, and it's starting with a bang--a big one courtesy of the Cabrillo Music Festival. The festival's 36th season, beginning Friday, Aug. 7, has the makings of a sonic thrill ride through musical territories that few--if any--of us have heard before. The high-octane repertoire spans the Americas, from the Bronx to Uruguay, and, most notably, is not for musical wimps.
Music director Marin Alsop, now in her seventh season with the festival, has an exuberant agenda planned that optimizes the festival orchestra's world-acclaimed virtuosities and draws from the compositional energies of some of today's brightest talents. Out of the 10 living composers represented in this six-concert run, only three--Richard Danielpour, Russell Peck and Joseph Schwantner--will not be in residence, which, if you do your math, leaves us with seven composers live and in the flesh. And a magnificent seven they are: Miguel Del Aguila, Christopher Caliendo, John Corigliano, Michael Daugherty, Michael Hersch, Christopher Rouse and Joan Tower. It's an amazing guest list, and auspicious too, considering that three of them--Corigliano, Rouse and Tower--are Pulitzer Prize winners.
Also slated are works by three late, great American composers, two of whom festival audiences have heard from quite regularly since Alsop's appointment-- Leonard Bernstein and Aaron Copland. The other is a most unlikely, but welcome, name: Frank Zappa, whose featured piece, The Perfect Stranger, pretty well summarizes his place in the scheme of Cabrillo things.
Alsop is especially pleased with this year's series, in part because of the pieces being performed, but also for what is being omitted.
"I'm excited that we're back to really focusing on the music," she says in a phone conversation from her home in Colorado. "After working in the past seasons with film, dance and opera, it's great to be emphasizing what we're here to do--to play music."
The festival opener, Michael Daugherty's Metropolis Symphony, adds an animated presence through witty interplays of Superman-based themes. The work's over-the-top hi-jinx and garish orchestral colorings have generated an avid following over the past two years. One suspects that the work's blend of humor and accessibility will make it a favorite in Santa Cruz as well.
"This piece allows us to enjoy ourselves; it's about serious listening and having fun at the same time," Alsop says.
Opening night then takes off at break-neck speed with Richard Danielpour's curtain-raiser, Toward the Splendid City. Danielpour is among the Eastern seaboard's most honored up-and-comers. His music is savored for its sonorous elegance and lyricism, but in this instance Alsop assures us we'll experience the composer's fire-breathing side. In terms of tonality, one might find it useful to simply analyze it on the Richter scale.
To offset all this pathos, Alsop has included Christopher Caliendo's lyrically inclined Trio Concertino, featuring Rachel Purkin, violin; David Young, double bass; and the composer himself on guitar. Alsop describes this new work--receiving its world premiere--as "flavored by strains of Argentinean and American folk music." (Caliendo and friends will return on Sunday night, joined by festival soloists, as well as dancers Natalia Mavor and Allen Walls, for a scintillating program titled "Tango Americano.")
Saturday night's bill, "Big Bang--Music for a Great City," evens out the decibels from Friday's opening blow-out with a repertoire of volatile modernity. The evening commences with Uruguayan composer Miguel Del Aguila's Conga-Line in Hell. "Pure madness," Alsop says. "I'd heard a terrific piece of his performed at Emily's [festival pianist Emily Wong] recital last year, so I asked him what he had for orchestra."
Daugherty's Motown Metal for brass and percussion alloys funk and fun in a way that arrests rockers right in their tracks, while Copland's no-less-adventuresome Music for a Great City (originally scored for the 1961 film Gone Wild) will stir memories for the more mature listeners who experienced the original jolt of the piece back in its heyday. Joseph Schwantner's Concerto for Percussion and Orchestra, with soloist John Kasica swinging the mallets, promises to be a spectacle to hear.
The complete CabMuFest schedule.
THE ENERGY CONTINUES at peak levels for the festival's second week. Both the Aug. 8 and 9 concerts trumpet 60th birthday salutes to composers Joan Tower and John Corigliano, respectively. On Saturday, the festival showcases three works by Tower, America's most celebrated woman composer: Fanfare for the Uncommon Woman (Aaron Copland's got nothing on this lady), Duets and Music for Cello and Orchestra, featuring the festival's principal cellist, Lee Duckles, as soloist.
Sharing the limelight with Tower will be a piece by the founding Mother of Invention, Frank Zappa. Zappa's The Perfect Stranger is one of many orchestral works he composed before his untimely death in 1993. Alsop has performed one of Zappa's pieces before (Duprez Paradise), but she confesses that The Perfect Stranger appeals to her even more. "I especially like this one," she says. "It has a storyline, you know. It's about a vacuum cleaner salesman, and at the beginning you hear these chimes as the doorbell. It's very amusing."
Also in store is the world premiere of a work by 26-year-old Michael Hersch titled Prelude and Fugue. "A work of Michael's won a competition held by Concordia [Alsop's N.Y.-based chamber orchestra] a couple years ago," notes Alsop. "It may be early to really tell how his personal style might develop, but he's very gifted."
The festival finale on Aug. 9 at the San Juan Bautista Mission will, as in recent years, be performed twice, enabling everybody to attend--if both performances don't sell out well in advance.
The mission's warm acoustic environment always provides a sympathetic atmosphere for the festival orchestra, and Corigliano's richly textured scores-- Fantasia on an Ostinato and The Red Violin-- are perfect choices. The latter, featuring violin soloist Brian Lewis, was scored for a film of the same name, due for release this fall. Fantasia..., according to Alsop, is Corigliano's single foray into the "minimalist" camp. Lewis will also perform in Bernstein's most ravishing orchestral work, Serenade (after Plato's Symposium). The concert's denouement will be Christopher Rouse's rousing Symphony No. 2, an assured way for this year's Cabrillo Music Festival to go out like it began, with a bang.
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Direct Hit: Cabrillo Music Festival director Marin Alsop leads the festival orchestra through an exercise in musical modernity at this year's concert series.
From the August 6-12, 1998 issue of Metro Santa Cruz.
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News and Announcements Archive
WSU developing efficient, cost-effective method for outsourcing large-scale data crunching and computer storage
DETROIT– As today's businesses struggle to meet their needs for large-scale information processing and storage, one Wayne State researcher is working to make outsourcing these services a more efficient, cost-effective option.
Song Jiang, assistant professor of electrical and computer engineering in the College of Engineering and resident of Troy, Mich., recently received a $400,000 Faculty Early Career Development (CAREER) Program award from the National Science Foundation to develop a more efficient method for quantifying Quality of Service (QoS), a guaranteed level of performance in data flow. The technology will be developed for large IT companies including Microsoft, Google, and Amazon.com, who offer their cloud computing and storage services with their extensive IT infrastructures to organizations looking for more computing power and data storage space.
Although a popular idea, outsource computing and virtual storage services have not developed an efficient method of quantifying hard disk input/output QoS – a parameter needed to predict the price for service. "Hard disks are mechanical, and the information on them is stored much like a record," Jiang said. "Depending on what information is needed, the time spent locating the information can vary. This inconsistency makes it difficult for companies that outsource their data storage and processing services to specify service quality and set a rate for services."
To solve the problem, Jiang is proposing the implementation of consolidated storage service based on a reference storage device, which can be the user's own system. The user is then guaranteed QoS that is as good as their own system, regardless of variations and changes in data access patterns.
Jiang's research is of high interest to IT giants such as Yahoo!, who use very large scale consolidated storage as an infrastructure for mission critical applications such as Yahoo search engines. For his research, Jiang is receiving technical support from Yahoo! as well as the Performance and Architecture Laboratory at Los Alamos National Laboratory, a United States Department of Energy national laboratory, and Seagate Research, a manufacturer of hard drives and storage solutions.
"As today's computing relies more and more on efficient and reliable access to large amounts of data, this research has the potential to significantly improve the productivity and efficiency of many IT services," Jiang said. "It will be important both for mid-size companies outsourcing their computing and virtual storage as well as very large organizations looking to build or expand their own computing facilities."
Jiang's research will also serve as a source for virtual data and storage system training for his student assistants and curriculum for WSU computer and electrical engineering students. "We're excited not only to pave the way for one of the most relevant technologies of the future, but also address the concern for the lack of strong data and storage system professionals in the IT industry."
# # #
Wayne State University is one of the nation's pre-eminent public research universities in an urban setting, ranking in the top 50 in R & D expenditures of all public universities by the National Science Foundation. Through its multidisciplinary approach to research and education, and its ongoing collaboration with government, industry and other institutions, the university seeks to enhance economic growth and improve the quality of life in the city of Detroit, state of Michigan and throughout the world. www.research.wayne.edu
For more information, contact Julie O'Connor at email@example.com or Amy Oprean at firstname.lastname@example.com.


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Tascam DR-07 Portable Digital Recorder videos
There are currently no videos for this product. If you are registered with us you may log in to add additional videos related to this product. You can also register an account to add a related video.Tascam DR-07 Portable Digital Recorder Reviews
Tascam DR-07
I chose the Tascam over other makes as I use a tascam multitracker as well and was happy with the brand quality. The unit does all it says it can and I have recorded 2 full band rehearsals so far. The auto level control isnt suitable for use in loud environments and was choppy but the manual setting when adjusted correctly was fine. It was set far too low in the first rehearsal but bringing the level up in sound forge by normalising it recovered the signal but didnt result in any excess noise appearing. Using 2000mA ni-mh rechargeable batteries didnt get the 7.5 hours battery life but more like 4 hours. The overall audio quality was good and the unit easy to use without too much referring to the manual. It would be nice to be able to skip through a track faster to find specific points as a 1 hour take took a lot of trawling through. Also it would be good to be able to rename your files after recording so you can put some form of identification to them. The driverless install is welcome and XP and Vista detected the external drives without problem. I used Sony Soundforge Pro 9 for post production. It would have been nice of Tascam to throw in an audio editor for those who dont posess one as some form of PP software is essential to get the best from the DR-07. (The Yamaha unit at the same price gives you a lite version of Cubase). Enough of the gripes - What you do get is an all in one pocket sized near studio quality recording soloution for less than the price of a decent mic. Nice touch to see a line in and separate mic input as well. Unfortunately our rehearsal room was far too bright and echoey, Had this not been the case some of the tracks captured would have been just in the frame to distrubute on a demo cd or post up on the web. The unit worked well with the \"Audacity\" freeware audio editor and with plenty of free burning software you\'ve no excuse! I bet Tascam would love to find a band that gets signed using one!


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James: This is precisely why I don;t have children: because their feelings always have to come first terribly inconvenient.
Ann: I'm not a mother. I'm ill-equipped, I'm fumbling all over the place. Don't even ask me where my commonsense went.
James: You lost it at, "brain tumor."
International Episode Titles:
Czech Republic: My o vlku (Speak of the Devil)
Original International Air Dates:
Czech Republic: February 6, 2011 on Prima COOL
Featured Music:
"Keep It Loose, Keep It Tight" by Amos Lee
"Peter and the Wolf" by Joseph Sitron
"Dressed To Kill" by Landon Pigg
"TNT" by Elliot Lawrence Big Band
"To Grandmother's House" by The Rabid Fans
This episode marked the series new time slot at 8:00 pm, rather than 9:00 pm.
In bocca al lupo is Italian for, "in the mouth of the wolf." It is actually used to wish a person good luck, whereupon the reply is, "May the wolf die!"
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Province blamed after surf tragedy Park cuts endanger lives, says searcher at Botanical Beach
Monday, December 02, 2002
Debra Brash, Times Colonist / Port Renfrew volunteer firefighter Greg Roy scans the turbulent waters off Botanical Beach Saturday for a 28-year-old Victoria hiker who was swept out to sea and is presumed to have drowned.
The provincial government is risking lives by withdrawing park ranger services at Botanical Beach provincial park, Port Renfrew volunteer firefighter Greg Roy said Sunday.
Roy was one of the emergency response crew called out Saturday morning to search for a 28-year-old Victoria man who was swept off the rocks by a thundering wave.
The man was hiking with a 26-year-old friend when an enormous wave rolled toward them. Despite the pounding surf, the younger man managed to swim back to shore and run for help. His friend was swept out to sea.
Sooke RCMP have not yet released the name of the victim who is missing and presumed drowned.
Roy believes keeping people safe on the dangerous West Coast is too big a job for his small town. "The provincial government started it. They put all this money into the park. They improved the road.
More and more people are coming here because they hear the surf is the thing to see. And now it's really putting pressure on our small community service and we don't have anything to go out there and get people who fall off the rocks," said Roy.
The volunteer firefighter wants park rangers working year round to explain to tourists how dangerous and unpredictable the ocean can be. He also wants better signs warning tourists to keep away from the lower rocks and tidal pools.
The provincial government took over the park about eight years ago. Since then, the spectacular marine park, like other provincial parks, has been affected by government funding cuts. Rangers patrol the 47-kilometre Juan de Fuca trail during the summer months. By mid-October, their work is finished.
Roy is concerned that Port Renfrew does not have a marine rescue service. After he heard the fire bell, he raced to the beach, scanning the horizon for signs of life. There was nothing else he could do. As he stood, with eight-metre waves crashing around him, he noticed a group of young women drinking on the rocky lower beach in the same place where the wave had knocked the two men off the rocks.
"I had to get them up to safer ground," said Roy. "They think it's safe and the next minute there's a tremendous surge."
If park rangers can't patrol the beach, Roy thinks the government should consider fencing off the tidal pools and the rocky areas during the winter. "People shouldn't be allowed to wander on the rocks," said Roy. "I went down to the tidal pools after the tide went out to look for the young man's body and I was still scared. I kept a watchful eye on the big waves."
Maurice Tremblay, a trustee who sits on Port Renfrew's local service committee, said since the provincial government took over the park, the number of visitors has increased from about 40 a year to 50,000 a year. "We are not equipped to go in the water. In fact, no one in their right mind would go in the water on a day like yesterday," said Tremblay. "Our situation is all voluntary. It's not really right that they ask volunteers to go there. Most of the time, it's a hopeless case. It's game over."
Rather than putting up better signs or hiring park rangers, Tremblay thinks the park should be closed during the winter. "I would say the whole West Coast is not a good place to come in the winter time. It's too dangerous."
Dick Heath, regional manager of Environmental Stewardship for the Ministry of Water, Land and Air Protection, said he would look into the circumstances of the accident. "We'll look at the level of signage and how effective the message is being communicated and if enhancements are necessary, we'll certainly bring them in," he said.
The government spokesman said he did not want to speculate on the need for year-round park rangers. "I'm not convinced having anyone on site would have made a meaningful difference," he said.
Fencing off or closing off areas is usually not effective in keeping people out of areas they are not supposed to be in when they are intent on visiting an area, said Heath. Since Roy moved to Port Renfrew 12 years ago, he has been called out to three or four drownings.
"It's a growing concern because more and more people are coming out here," he said.
© Copyright 2002 Times Colonist (Victoria)


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Whether you're whale watching, walking on a volcano or relishing miles of white beaches, you'll find your paradise in Hawaii. As a guest at any of our Hawaii Ascend Hotel Collection hotels, we want to ensure that you find rooms as welcoming as The Aloha State's heavenly weather — while offering high standards of luxury and the true local flavor you crave.
Elevate your viewpoint by hiking at Diamond Head or Hawaii Volcanoes National Park. Splash through miles of coastline at Waikiki or Hanauma Bay. Pick a pineapple at the Dole Plantation and have a truly Hawaiian experience at the Polynesian Cultural Center. Relive the "day of infamy" at the USS Arizona Memorial.
Let the Destination Reach You at any of our Ascend Hotel Collection Hawaii hotels, whether you're looking for business or tourist destinations. Enjoy the amenities of a luxury resort, with the intimacy of a bed and breakfast. Most of our rooms contain cable television, free high-speed Internet access and mini bars. Book a room at any of our HI Ascend Hotel Collection hotels, listed below.
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Grand Central Theatre
33 Grand Central Terminal,
New York,
5 people favorited this theater
This little Art Moderne-style newsreel theater operated inside the vast Grand Central Terminal from 1937 until about three decades later, when the space was gutted for retail use. The theater also screened cartoons and short subjects. A large incandescent clock above the screen reminded theater-goers of the time. At the rear of the auditorium was a standing-room section, and a small bar was located near the theater's entrance.
It was located near track 17 and today the area is used as a wine shop, complete with the beautifully restored star-covered ceiling of the former theater's lobby.
Recent comments (view all 11 comments)
I think I remember reading that later on it screened second run fims.
Where can I get a better copy of this postcard? I would like to use it in my research
When I was a regular customer in the early '50s, they changed the program twice a week (to run current news in that pre-TV era). Since the change-over came in the middle of the day, and if you timed it right, you could catch two programs (a two-hour show) for a single admission ($1).
Some other newsreel and short subject cinemas located in train stations:
South Station Theatre, Boston
Newsreel Theatre, Cincinnati
Victoria Station News Theatre, London
Hi Joe Clark & my other theater enthusiast friends!!! Every so often, you can find a Grand Central Theatre postcard on Ebay.
Though it won't really map properly, I believe the address should be listed as 33 Grand Central Terminal (Main Concourse, opposite Track 17), New York, NY 10017.
a short video of people going into the grand central theater is on the video VHS titled: "The winning of world war 2" "The road to victory – ratling the saber" 1990 mbi/ulanoff distributed by publishers choice videos. hosted with personal commentary by John s. d. elsenhower. I own this video.
bill gilbert
The April 1, 1937, issue of Motion Picture Daily said that the opening of the newsreel theater being built inside Grand Central Station had been set for May 3.
I passed by it many times but never went in. (In the early 1970's, when I was going to college and Amtrak trains on the old New York Central lines were still leaving from Grand Central, I had to change stations in New York.) I distinctly recall that on one occasion, oe of the last times I saw the theater in operation, the program was the 1971 documentary "The Hellstrom Chronicle", so the end must have come around 1973 or thereabouts.
Another thing: it probably was an inspiration for a strip in the comic series "Julis Knipl: Real Estate Photographer", which takes place in such a theater.
Just login to your account and subscribe to this theater


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Hang tight!
Recipe by namiknows
"A quick, light couscous stir-fry with Indian flavors. Great for vegetarians, but easily adapted into a chicken dish. Feel free to substitute veggies any way you like. Other options include sliced zucchini, string beans, eggplant, carrots, snow peas, and celery. Also try adding sesame seeds, flax seeds, or sesame seeds towards the end of stir-frying. Can be served with plain yogurt."
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1 cup vegetable broth
1/2 cup couscous
1 tablespoon canola oil
1 teaspoon ajwain (carom) seeds
1 clove garlic, coarsely chopped
1 teaspoon turmeric powder
1 teaspoon curry powder
1/2 teaspoon hing powder (asafoetida)
1 pinch chili powder (optional)
1 pinch ground cinnamon
1 (12 ounce) package frozen shelled edamame (green soybeans)
1 bunch broccoli, cut into florets
sea salt to taste
ground black pepper to taste
1 tablespoon toasted sesame oil
This was very tasty and pretty easy to make as well. I loved the blend of spices. As a carnivore, I decided to add chicken. With the chicken, it probably does not need as much edamame as the original recipe calls for (there was a lot of edamame!) Yum!!
This was just ok.
4 Ratings
This turned out really really good! I used zucchini instead of broccoli and peas instead of edamame, but that is just because it is what we had on hand. A keeper!
* Percent Daily Values are based on a 2,000 calorie diet.
Indian Curry Couscous with Broccoli and Edamame
Serving Size: 1/3 of a recipe Servings Per Recipe: 3 Amount Per Serving Calories: 413 Calories from Fat: 162
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Chicken breasts simmer in a spicy tomato and yogurt sauce.
See how to make a quick-and-easy, Indian-inspired seafood curry.
Tasty white fish fillets are baked in a spicy curry sauce.


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Posted in December 2010
Domain Name Registrar Agrees to Crack Down on Web Sites Selling Prescription Medications Unlawfully
Topics: diversion, Internet pharmacies, and NABP
Potentially thousands of rogue Internet drug outlets will have to board up their virtual storefronts, thanks to a new partnership agreement enforcing a major domain name registrar's ban on illegal use of the Web sites it hosts.
CS Prescriptions for Teens Increase to Nearly Twice 1994 Levels
Topics: Teens, Prescription drug abuse, and NABP
One in nine adolescents received a prescription for medications with a high potential for abuse in 2007, a rate almost twice the 1994 levels.
Read more →


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Faulkner, Dorothy and Coates, Elizabeth
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URL: | http://www.routledge.com/books/details/97804155656... |
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This chapter introduces the edited collection, Exploring Children's Creative Narratives that offers fresh perspectives on children's creative narratives and that explores what these perspectives reveal about their imagination, their thought processes and how they understand the world. The chapter argues that ethnographic case studies, naturalistic observations, conversations and playful interactions with children ranging in age from two to eleven years allow the development of theoretical insights that challenge traditional accounts of creativity and narrative. It also argues that a proper understanding of creative narratives has to be an interdisciplinary endeavour if it is to do justice to the rich, complex, multi-modal and embodied nature of the children's thought processes as revealed through their drawing and story-telling, music making, dance, drama and imaginative play. Interpretation of these processes draws on socio-cultural accounts of creativity and creative development that challenge more traditional accounts of creativity as an attribute or talent that belongs only to certain gifted individuals. The chapter (and contributors to the collection) maintains that to understand the situated nature of children's creative activity it is important to examine the social, affective and cognitive processes that take place when children are immersed in such activity. This re-conceptualisation of children's creativity challenges conventional educational practice and suggests that formal educational training programmes should offer teachers more sophisticated cultural discourses and experiences which will allow them to gain a more rounded understanding of children's creative narratives.
Item Type: | Book Chapter |
Copyright Holders: | 2011 Dorothy Faulkner and Elizabeth Coates |
Keywords: | children's creativity; children's narratives; creativity theory; creative education; early years |
Academic Unit/Department: | Education and Language Studies > Childhood, Development and Learning |
Interdisciplinary Research Centre: | Centre for Research in Education and Educational Technology (CREET) |
Item ID: | 28573 |
Depositing User: | Dorothy Faulkner |
Date Deposited: | 21 Apr 2011 09:21 |
Last Modified: | 27 Oct 2012 11:51 |
URI: | http://oro.open.ac.uk/id/eprint/28573 |
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Lab Equipment
Lab Benches Lab Chairs Lab Carts Tables/Clean tables Gowning benches Gowning racks Storage units Wire Shelving/Carts |
Cross-over ladders Dock ladders Rolling Safety Ladders Step Stools Step Ladders Stock picking ladders |
Lifting Equipment
Cargo Lifts Pallet Jacks Lift tables & carts Dock lifts Scissor lifts Stackers Tilters Vertical lifts (VRC) Work Positioners |
Loading Dock Equipment
Dock Boards Dock Bumpers Dock Levelers Dock Lights Dock Plates Dock Seals Dock Shelters Ramps Scissor Dock Lifts Strip Doors Wheel Chocks |
Single & Multiple Tier Steel, Plastic, Wood Locker Benches Trim & accessories Wire Storage Lockers |
Mailroom Furniture
Carts Containers Sorters Tables |
Anti-Fatigue Conductive Drainage Entrance matting Logo Mats Safety Matting |
Partitions/Wall Systems & Strip Doors
Air Curtains Curtain Enclosures Modular Office Systems Modular Wall Systems Preassembled Buildings Strip Doors Thermal Barriers Wire Partitions Wire Safety Barriers Wire Security Units |
Bar/Rod & Sheet Stock Bulk Cantilever Carton Flow Drive-in Pallet Rack (selective) Pipe Rack Stacking Racks Wide Span Wire Decks & Dividers |
Safety Equipment
Cabinets Guard Rail Systems Protective Barriers Rack Protectors Signs Speed Bumps Spill Containment Traffic Mirrors |
Industrial, Clip type Mobile Shelving Open Filing Plastic Shelving Records/Archive Steel Shelving Wall Mounted Wire Shelving & Carts |
Cafeteria Folding Lab Tables Machine Tables/stands Mobile Tables Shop Tables Utility Tables |
Truck Restraints
Load Bars Decking Beams Cargo Straps Bungee Cords |
Appliance Hand Trucks Bar Cradle Box Bulk Convertible Drum Hand Trucks Platform Trucks |
Deluxe Systems proudly serves Massachusetts, MA, New Hampshire, NH, Rhode Island, RI, Maine, ME, Connecticut, CT, Vermont, VT