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The Somerville Poet Laureate--It is finally here!
Harris Gardner (Tapestry of Voices) and myself (Doug Holder--Ibbetson Street Press) met with Gregory Jenkins,( The Director of the Somerville Arts Council), at the now defunct Sherman Cafe in Union Square this summer to discuss the prospects of getting a Somerville Poet Laureate. I have been pushing this for years, but to no avail. There were either vacant promises from local pols, the eye-rolling, the patronizing hand shake--slippery as a snake--well you know the drill. Jenkins was on board with the idea; so then we met with the mayor and he thought it was a good idea as well. Now we have an official announcement, and information about how to apply. We are forming a selection committee, so far it is Greg Jenkins, Doug Holder, Bert Stern, Harris Gardner, Linda Conte, Charles Coe, Ifeanyi Menkiti and others. So if you are a fine poet, community-minded, have a track record of promoting poetry, and have a strong vision for your possible tenure--apply!
Application and Overview
Statement of Purpose
The City of Somerville announces the creation of a Poet Laureate for Somerville. The City views the position as a means to further enhance the profile of poets and poetry in the city and beyond. The Poet Laureate is expected to bring poetry to segments of Somerville's community that have less access or exposure to poetry: senior citizens, youth, schools and communities. The Poet Laureate will be a person of vision with the ability to enact his/her vision.
The Poet Laureate will serve for a two-year term and will be provided an honorarium of $2,000 per year. A contract will be derived with expectations detailed as to the public benefit required of the position, which will be jointly determined with the final applicant and review committee. The expectation is that the position will support and expand poetry in the city. The Somerville Arts Council/City of Somerville will support the Laureate in networking within the community but actual work must be accomplished by the chosen candidate.
How to apply
Deadline: Postmarked by November 17, 2014
Deadline: Postmarked by November 17, 2014
Candidates for Somerville Poet Laureate must provide the following:
- One page contact info sheet with name, address, phone number, email, website (if applicable)
- Proof of residence demonstrated by sending a copy of a utility bill, lease, phone bill. (a jpg image of a current bill or statement is fine if emailing application, or a photocopy of statement if mailing application)
- Curriculum Vitae / Poetry-Related Bio
- Up to 20 pages of original poetry
- One to three-page vision statement with details as to how you will implement the public benefit component.
How to submit
- Either email PDFs of the above items to Gregory Jenkins at email@example.com with Poet Laureate in the subject header:
- Or mail the following documents to: Somerville Poet Laureate, Somerville Arts Council, 50 Evergreen Ave., Somerville, MA 02145
Selection Process for Poet Laureate of Somerville
A committee, comprised of local poets, teachers, and arts administrators, will review the applications based on the evaluation criteria and select no fewer than three and no more than six applications to be finalists. Finalists will be interviewed in December with the expectation that they will further refine their proposed vision and public component for the position. The interview process will also provide the selection committee the ability to inquire more of the candidate. Based on the four criteria below, the committee will select a final candidate and alternate who will be presented to Mayor Joseph Curtatone for his approval.
Evaluation Process for Poet Laureate Nomination
The Poet Laureate will be reviewed and chosen on the basis of the four criteria (percentage weights included):
· Excellence in craftsmanship, as demonstrated by submitted original poems (25%)
· Providing a vision for the position. How will you work with the community, schools, nonprofit or municipal arts and service departments. Please convey your vision for the position with details of outreach and collaborations. (25%)
· Professional achievement in the field of poetry. Merit shall be proven by publication credits either in small press or large press publications; at least one collection, full size or chapbook published by a small press or large press; also, awards or recognition such as grants, fellowships, prizes, and/or other recognition. (25%)
· A history of actively promulgating the visibility of poetry in Somerville's neighborhoods and literary communities through readings, publications, promotion of events, public presentations and/or workshops and other types of teaching and literary community involvement. (25%)
City of Somerville
Mayor Joseph A. Curtatone


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subset: commoncrawl


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1920 - 2017
Captain Charles 'Chic' Eather (Ret.)
Charles 'Chic' Eather was born on August 2 1920 in Sydney Australia. His first job was with a local firm of stove manufacturers. He hated the work!
In 1940, he became an anti-aircraft gunner and was discharged whilst waiting for his Air Force call-up. In the interim he joined the Merchant Navy and took his anti-aircraft friends to Suez. While on the high seas he missed his RAAF call-up and was removed from the list. With the money he saved as a Merchant Mariner he completed his flying training (that had begun in 1936) at the Royal Aero Club, Mascot, becoming a first-officer with Ansett Airways and concurrently commissioned a Flying Officer with the Citizens Air Force Reserve.
At war's end (1946) he joined the fledgling Roy Farrell Export-Import Company, the forerunner of Cathay Pacific Airways. He flew during the Karen Insurgency in Burma for the Union of Burma Airways. In 1953 he returned to Cathay Pacific Airways and stayed with them until his retirement in 1975, amassing 25,000 flying hours.
Chic retired to the Queensland Gold Coast with his beloved Chinese wife Judy. They have two children.


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Kristin has been a nurse since 2000 and has worked in a variety of nursing settings. The majority of her nursing career has been spent working in post-anesthesia care units and outpatient surgical centers. She routinely cares for patients who have undergone myringotomies (ear tube placement), tonsillectomies, nasal sinus surgery and other ENT procedures. Working closely with ENT physicians, she not only cares for these patients, but educates them about their condition and teaches them how to care for themselves at home.
Kristin received an Associate Degree of Science in nursing and graduated with honors from Weber State University in 2002. Prior to that she had earned her Practical Nursing certification through Utah Valley University in and became a Licensed Practical Nurse in 2000. Kristin also holds several certifications from the American Heart Association including: Basic Life Support, Advanced Life Support, and Pediatric Advanced Life Support.
ENT disorders are among the most common health problems diagnosed in both adults and children. As a survivor of thyroid cancer, I know first hand how debilitating and frightening any illness can be. No matter what your specific health problem is, education is both comforting and essential. I hope that others will use my site to learn valuable information to improve their health and get the care that they need.


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"Facts are stupid things."
I started out to post a blog about misquotes. So using the trusty Quotations Page, I searched for a quote about misquotes. The above sentence was spoken by Ronald Reagan in 1988. Ironically, it was a misquote of John Adams who said "Facts are stubborn things." A misquote of a quote about misquotes. Or a wrong fact about facts. This will be long. Hopefully a few of you will struggle through it.
Recently, Rudy Giuliani supposedly warned of another 9/11 if the Democrats win the White House in 2008. In reality, he did not say that. When reading what he really said, he warned that the Democrats would take the US to a defensive position, the same approach that was taken before 9/11. Even reading the article at politico.com, his quotes don't match the headline. While I don't agree with all that Rudy says, and I think he generalizes WAY too much, he's on target with what he says about the approach to the war. (I don't believe all Democrats feel the way he says or that all Republicans do either)
A lot of people have said that Rudy G. politicized the deaths of 2,973 people. I don't see it that way. I read the quotes and think he's politicizing the potential death of 2,973 +/- future people. He's talking about preventing the NEXT 9/11. You may agree with his points and you may disagree with his points. But be sure you talk about his points, not what he supposedly said.
This reminds me of Murphy Brown. In 1992, Dan Quayle gave a speech about families in America. Excerpts are posted here. He was ridiculed by the press, by late night comedians, by other candidates. Trouble was, once they read what he said, they all AGREED with him!!! After all was said and done, I wrote a letter to the editor (no blogs were around then) that asked newspapers to publish speeches in entirety.
With the internet, there's little excuse not to read a candidate's speech before commenting on a headline. Politico did a lousy job of reporting. I don't believe, this is a liberal conspiracy, I don't believe in that any more than I do a right-wing conspiracy. I believe it's a simple attempt to sell newspaper or web hits.


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Visit TwiML Apps in the Twilio Console and click 'Create TwiML App'. Enter 'Mobile Quickstart' as the app's Friendly Name and click 'Save'. Don't worry about the Voice and Messaging URLs yet – we'll fill them in shortly.
Once the application is saved, make note of its ApplicationSid (a long string like "APa4983020a0b29fb5db1b1dc68e322fbe"). We'll need this in the upcoming steps.
Next, we'll deploy a server to perform two functions:
The code for the Quickstart Tutorial server application can be found in the
Mobile Quickstart repository that
we downloaded in the previous step. You may want to open
server.py and take a
look. The app uses the popular Flask framework and
exposes three endpoints:
'/' generates TwiML which tells Twilio to simply echo "Welcome to Twilio" - we will use this to verify that everything is wired up correctly.
'/call' generates TwiML which can route a call between the phone network and your Android application.
'/token' returns a Capability Token to your Android application.
Now, we need to make this application accessible via the public Internet. Why? Because Twilio's servers (which are connected to the phone network) need to access your server in order to fetch instructions for how your Twilio calls should be handled. This is a standard requirement for any Twilio-powered application. You can do this with either of the following methods, or use another approach that you prefer.
One of the easiest ways to deploy the application is using Heroku. To do this:
Config Variables.
Once Heroku finishes, launch a second tab and return to the configuration screen for your TwiML app. Enter your Heroku application's base URL as the Voice Request URL for your application and save the change:
Request URL: https://bosco-baracus-1201.herokuapp.com/
With this method, you'll run
Flask on your local machine using python, then
create a publicly-addressable 'secure tunnel' to it using ngrok.
server.py and modify
ACCOUNT_SID and
AUTH_TOKEN to match the values from your Twilio account.
APP_SID to match the App Sid you created above.
CALLER_ID to any caller ID that you've verified with your Twilio account. You can verify a new Caller ID here. Alternatively, can also use any Twilio Phone Number that you've purchased.
pip install -r requirements.txt from the
Server/ directory at the command line.
python server.py to start the server.
ngrok http 5000 to open up a tunnel from the public internet to your server.
Return to the configuration screen for your TwiML app. Enter your ngrok base URL as the Voice Request URL for your application and save the change:
Request URL: https://d75ca7cc.ngrok.io/
To make sure your server deployed successfully, open a web browser and browse to the url
view-source:{your heroku app or ngrok address}/. If everything is working, you'll see some XML that looks like this:
<?xml version="1.0" encoding="UTF-8"?> <Response> <Say>Welcome to Twilio</Say> </Response>
This is a simple TwiML application that will play an announcement whenever you make a call from your Android application.
Now that we have the server-side dependencies setup, let's get started on our Android app. You can test the examples below in the Android Emulator or on your device.
We all do sometimes; code is hard. Get help now from our support team, or lean on the wisdom of the crowd browsing the Twilio tag on Stack Overflow.


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Make your own magnetic decals with our magnetic inkjet printable decal paper. All you have to do is print your image on any inkjet printer. This is a non-adhesive material that uses magnetism to adhere to metal surfaces. Material is approximately 11 mils thick, has a surface magnetism of about 90 gauss with a magnet thickness of about 8 mils. It sticks well to any clean metal surface. You can cut this magnetic decal paper to any size or shape after you run it through your standard desktop inkjet printer.
Copyright ©
Built with Volusion


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Studies of low basal Jun N‐terminal kinase (JNK) activity in non‐stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S‐transferase Pi (GSTp) and was characterized as a JNK‐associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp–JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun–JNK complex caused a dose‐dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp‐derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c‐Jun ubiquitination and decreased c‐Jun‐mediated transcription. Co‐transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1–MKK4 module. Mouse embryo fibroblasts from GSTp‐null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases.
Jun N‐terminal kinases (JNKs) belong to the multi‐member family of stress kinases that are activated transiently in response to UV‐ or X‐irradiation, heat shock, osmotic shock or inflammatory cytokines (Galcheva‐Gargova et al., 1994; Kyriakis et al., 1994; Westwick et al., 1994). JNK activation in response to UV irradiation is mediated by upstream signaling components, including cdc42, p21PAK, ASK1, MLK, MEKK1, SEK1/MKK4, MKK7 (Coso et al., 1995; Fanger et al., 1997; Tournier et al., 1997; reviewed by Ip and Davis, 1998) and p21ras (Minden et al., 1994; Adler et al., 1995a, 1996), in concert with nuclear DNA lesions (Adler et al., 1995b). Different forms of stress have been shown to mediate JNK activation via various cellular pathways (Adler et al., 1995c). Activated JNK phosphorylates the transcription factors c‐Jun, ATF2, p53 and ELK‐1 (Kyriakis et al., 1994; Gupta et al., 1995; Whitmarsh et al., 1995; Adler et al., 1997; Fuchs et al., 1998a). Phosphorylation by JNK has been implicated in stabilization (Fuchs et al., 1997, 1998b) and transcriptional activity (Kyriakis et al., 1994) of its substrates, which directly contribute to the mammalian stress response through changes in the cell cycle, DNA repair or apoptosis (Xia et al., 1995; Fuchs et al., 1998a; Kasibhatla et al., 1998).
Despite the significant advances in identifying the components of stress‐activated protein kinases, the mechanisms underlying the regulation of JNK before and immediately after stress are not well understood. In cells maintained under normal growth conditions, the basal activity of JNK is low, although JNK phosphorylation by upstream kinases occurs in response to growth factors (Minden et al., 1994) and hence should also be observed in cells proliferating under normal growth conditions. While some unstressed cell types have been found to contain high levels of JNK activity (Dhar et al., 1996), the reason for this high basal level is unknown. A 2‐ to 4‐fold increase in constitutive JNK activity has been reported and, while this is lower than the activation upon exposure to DNA‐damaging agents, it is equivalent to tumor necrosis factor‐α (TNF‐α) treatment and the degree of activation elicited by multiple exposures to low dose UV‐B (Adler et al., 1995d). As a key component in regulating the stability and activity of its substrates (Fuchs et al., 1998c; Kyriakis et al., 1994), changes in JNK's basal activity could influence key cellular functions, including growth, apoptosis and transformation.
Neither transcript nor protein levels of JNK are affected by stress. JNK activation is accomplished upon its phosphorylation by upstream kinases in response to stress (Coso et al., 1995; Tournier et al., 1997). Dephosphorylation of JNK at a later stage (Cavigelli et al., 1996; Hanada et al., 1998) is thought to constitute the primary mechanism underlying the regulation of JNK activity as a kinase. Nevertheless, several studies have demonstrated the existence of JNK inhibitors in normal growing cells. Thus, stress‐mediated JNK activation may be independent of its upstream kinases. Among the cellular components involved in regulating JNK substrates is a 'δ inhibitor' which blocks transactivation of c‐Jun by interacting with the δ domain (Baichwal et al., 1991). This is an integral 27 amino acid sequence (30–57) from the N‐terminal region of c‐Jun required for JNK binding to c‐Jun, enabling Jun phosphorylation (Adler et al., 1992) or ubiquitination (Treier et al., 1994; Fuchs et al., 1996). Other JNK regulatory proteins include p21cip/waf1, which has been shown to exert greater inhibition of JNK activity in response to stress (Shim et al., 1996).
Our interest in exploring the nature of JNK's low basal activity originated in the observation that extracts of non‐stressed cells efficiently inhibited c‐Jun phosphorylation when added to a solid‐phase kinase reaction. Our further studies led to the purification, identification and characterization of glutathione S‐tranferase Pi (GSTp) as a JNK inhibitor, described in this report.
GSTs comprise a multigene family, of which GSTp is the most prevalent and ubiquitous non‐hepatic isozyme (Jakoby, 1978). Among cellular functions attributed to GSTs are ligand binding and xenobiotic detoxification (Tew, 1994). Reduced glutathione (GSH) binds to the 'G' site of GSTp (and other GST isozymes) and plays an important role in detoxification of reactive oxygen species (ROS) and the maintenance of the cellular redox state (Sato et al., 1989).
Among factors implicated in regulating JNK activity are ROS and altered redox potential (Adler et al., 1995c; Gomez‐del‐Arco et al., 1996; Cui et al., 1997; Wilhelm et al., 1997). ROS have also been associated with regulation of other signaling cascades, e.g. certain isozymes of protein kinase C (PKC; Konisshi et al., 1997) and mitogen‐activated protein kinase (MAPK; Guyton et al., 1996). The addition of exogenous oxidants or anti‐oxidants has been found to influence the activation of MAPK/JNK (Lo et al., 1996; Wilhelm et al., 1997).
Changes in ROS can also directly influence transcriptional activity, as demonstrated for NF‐κB (Wang et al., 1995), Ref‐1 (Xanthoudakis et al., 1992) and c‐Jun (Gomez‐del‐Arco et al., 1996). Thus, evidence is accumulating that redox status can play an integral role in kinase‐mediated stress response pathways. Our characterization of GSTp as an endogenous regulator of JNK activity provides a novel function for a protein with previously characterized catalytic and ligand‐binding properties.
A JNK inhibitor is present in cellular extracts of normally growing cells
JNK activities were measured in solid‐phase kinase reactions using Jun as a substrate. Proteins prepared from UV‐treated 3T3‐4A mouse fibroblast cells exhibited marked phosphorylation of c‐Jun. The kinetics of JNK activation in these cells increased within 1–5 min, reaching peak levels within 20 min and declining to basal levels within 3–4 h (Adler et al., 1995b). This is a transient activation that is monitored as a multifold increase of kinase activity relative to the control (untreated) cells. Since the assays are performed under conditions that enable selective binding of JNK isozymes (Adler et al., 1995b), we refer to the Jun‐bound kinase as JNK and to the overall complex as Jun–JNK.
When cellular extracts prepared from unstressed cells were added to the pre‐formed Jun–JNK complex before adding [γ‐32P]ATP, c‐Jun phosphorylation was inhibited by 80% (Figure 1A, compare lanes 1 and 2). To elucidate further the nature of this inhibition, proteins derived from the non‐stressed cells were absorbed by pre‐incubation with increasing amounts of Jun–JNK. Subsequently, beads‐bound c‐Jun–JNK complexes were spun and the supernatant, which lacked Jun–JNK‐bound proteins, was tested to determine its ability to alter the degree of JNK‐mediated Jun phosphorylation. Increasing the amounts of Jun–JNK complexes used to absorb the putative inhibitor resulted in a dose‐dependent decrease of inhibitory activity (Figure 1A, lanes 3–6). To determine whether this inhibitor activity was heat stable, the protein fraction that contained the inhibitor activity was heated to 95°C for 5 min. This treatment abolished the fraction's ability to block Jun phosphorylation by JNKs (not shown).
UV irradiation abolishes JNK inhibitor activities
The presence of JNK inhibitory activity in non‐stressed cells prompted us to determine possible changes to the inhibitory activity after UV irradiation. As shown in Figure 1B, extracts prepared after UV treatment lacked inhibitory activity. The decrease in JNK inhibitor activity depended on the UV dose. Whereas a 10 J/m2 dose (a 6 s exposure) caused a 40% reduction (as quantified using a phosphoimager), a 40 J/m2 dose (a 25 s exposure) completely abolished the inhibition. In all instances, analysis was performed using the same amount (20 μg) of protein prepared 30 min after administering UV irradiation (Figure 1B). A correlation between JNK activation and the inactivation of its inhibitory activity was also noted in human melanoma cells, which require a 60 J/m2 dose for JNK activation. In melanoma cells, 40 J/m2 doses caused a 50% inhibition, whereas a 60 J/m2 dose abolished JNK inhibitory activities (not shown).
Purification of the JNK inhibitor
To purify the putative inhibitor, extracts of normally growing cells were subjected to the six steps described below. After each step, the presence of the putative inhibitor (tested by measuring inhibition of c‐Jun phosphorylation) was monitored in a solid‐phase kinase assay using the hisJun–JNK (1 μg) complex. Proteins from non‐stressed cells were first precipitated in ammonium sulfate (50%). After dialysis against kinase buffer, the non‐precipitated proteins were concentrated on membranes with various molecular weight cut‐offs. Inhibitor activity was found in the flow‐through of both 100 and 30 kDa cut‐off filters; it was retained on the 10 kDa membrane, indicating that the inhibitor is between 10 and 30 kDa in molecular weight (Figure 1C). The 10–30 kDa fractions were loaded on a gel filtration column; several fractions within the 20–25 kDa range (based on calibration of the gel filtration column with known molecular weight standards) retained inhibitory activity (Figure 1D). Active fractions were pooled and fractionated on a MonoQ anion exchange column; a 70 mM NaCl eluate was found to contain most of the inhibitor activity (Figure 1E). Active MonoQ fractions were then loaded onto a phenyl‐Sepharose column in the presence of 0.6 M ammonium sulfate. Using decreased concentrations of ammonium sulfate, the inhibitor activity was found in 0.2 M eluate fractions (not shown). The active fractions from the phenyl‐Sepharose column were incubated in a hisJun–JNK column. Analysis of the hisJun–JNK‐bound material revealed a single band on silver‐stained gels (Figure 1F). Microsequencing analysis of the corresponding band obtained after a large‐scale purification (Table I) revealed a 12 amino acid peptide (Pro‐Pro‐Tyr‐Thr‐Val‐Val‐Tyr‐Phe‐Pro‐Val‐Arg‐Gly) that exhibited 100% homology to the human form of GSTp. To confirm this protein's identity, a purified form of GSTp was separated parallel to the hisJun–JNK‐bound material and subjected to Western blot analysis using polyclonal antibodies to GST. Both migration and immunoreactivity of purified GSTp were identical to those of the hisJun–JNK‐bound material (Figure 1F).
GST inhibition of JNK activity does not alter phosphorylation of Jun or JNK
To determine whether GSTp affected the degree of JNK or Jun phosphorylation, increasing concentrations of GSTp (purified form) were added to pre‐formed Jun–JNK complex which contained the phosphorylated form of JNK obtained from UV‐treated cells. GSTp decreased JNK phosphorylation of c‐Jun in a dose‐dependent manner (within a range of 0.05–1 μg) (Figure 2A), but it did not decrease the number of phosphate groups on JNK, as revealed by immunoblots with phospho‐JNK antibodies (Figure 2B). Dual activity protein phosphatase (Ishibashi et al., 1992) was used as a positive control in these reactions (Figure 2C).
To determine whether GSTp affects the number of phospho groups on c‐Jun, cellular extracts from normally growing cells were incubated with pre‐formed Jun–JNK complex for the indicated periods of time before or after the phosphorylation step with [γ‐32P]ATP. The extent of c‐Jun phosphorylation was not altered when cell extracts were added after the phosphorylation reaction (Figure 2D). This observation suggested that GSTp did not reduce the number of phospho groups on c‐Jun.
Effect of different GST isoforms on JNK activity
Incubation of whole‐cell extract prepared from non‐stressed mouse fibroblasts with the hisJun–JNK complex identified GSTp as the associated protein. A marked decrease in this association was found in proteins prepared after UV irradiation (Figure 3A). In addition to GSTp, isozymes of the GSTα and GSTμ families were also capable of associating with the Jun–JNK complex in vitro (Figure 3A). GSTp exhibited greater JNK inhibitory activity than did GSTμ, which was more potent than GSTα (Figure 3B). Bacterially expressed GST (GST‐2T) also mediated JNK inhibition (Figure 3B). This excludes the possibility that the inhibitor activity was dependent on any putative GST‐associated cellular component.
Dose‐dependent inhibition of JNK by GSTp
GSTp inhibition of JNK activities was determined using six concentrations of purified GSTp added to either full‐length or N‐terminal forms of c‐Jun. The addition of purified GSTp to JNK complexed with GST–Jun5–89 or hisJunfull‐length led to a concentration‐dependent inhibition of c‐Jun phosphorylation (Figure 3C). The greater inhibitory capacity of GSTp observed for GST–Jun5–89‐ compared with hisJunfull‐length‐based JNK complexes can be attributed to the different conformations of the recombinant substrates (N‐terminal GST–Jun5–89 versus hisJunfull‐length). A higher degree of inhibition of the GST–Jun5–89 fusion protein is not likely to occur as a result of GST–GST interactions, which would require other experimental conditions and result in different kinetics. Nevertheless, using either GST–Jun5–89 or hisJunfull‐length as substrates, the degree of GSTp inhibitory activity appeared to be linear within the range of 25–180 ng of GSTp (per microgram of pre‐formed Jun–JNK complex; Figure 3C). GSTp also inhibited JNK activity when added to a soluble form of hisJun–JNK complex (not shown).
Specificity of GST inhibition
To determine the specificity of the JNK inhibitor, purified GSTp was incubated with substrates for other protein kinases. Incubation with PKA, PKC, casein kinase II (CKII) or MAPK revealed 6, 3, 36 and 9% inhibition, respectively. Under the same conditions, GST elicited 86% inhibition of JNK (Figure 3D). In all cases, the activities of the various kinases tested were normalized (c.p.m./μg protein and an equal ratio between the respective substrate and GSTp) to ensure an equal degree of substrate phosphorylation.
GSTp immunodepletion attenuates JNK inhibition
We further elucidated the ability of GSTp to inhibit JNK activity by modulating its levels or activity, or both, in vitro and in vivo. The in vitro kinase assay in which proteins from non‐stressed cells were added to the pre‐formed Jun–JNK complex revealed a dose‐dependent increase in the degree of JNK inhibition (of up to 80%). Immunodepletion of GST from whole‐cell extract decreased the degree of JNK inhibition from 80 to 45% (Figure 3E, lane GST‐1). Repeated immunodepletion by GST antibodies further decreased the degree of JNK inhibition to 20% (Figure 3E, lane GST‐2). Control immunodepletion reactions with normal rabbit serum (NRS) and protein A/G beads did not affect the degree of JNK inhibition (Figure 3E, lanes NRS‐1 and NRS‐2, respectively).
Specific GSTp inhibitors and a GSTp‐derived peptide efficiently alter JNK inhibition
As an independent approach to inhibit GSTp, we used specific inhibitors which were shown to inactivate GSTp enzymatic activity in vitro and in vivo (Flatgaard et al., 1993). Adding TER‐117, a specific in vitro GSTp inhibitor, to a Jun–JNK complex at the same time as GSTp prevented GSTp inhibition of JNK activity. Similarly, TER‐293, another GSH peptidomimetic related to TER‐117, also blocked GSTp inhibition of JNK activity (Figure 3F). TER‐317, structurally similar to TER‐117, yet void of GSTp inhibitory properties, failed to affect GSTp inhibition of JNK. Similarly, TER‐199, an inactive prodrug of TER‐117 (Flatgaard et al., 1993), was also without effect in vitro.
Molecular dynamic calculations on the energyminimized X‐ray crystal structure of GST were performed to identify the most flexible regions of the protein, i.e. those that were computed to have the highest degree of fluctuations. These regions are most likely to undergo significant conformational changes during catalysis or ligand binding. Two such flexible domains, corresponding respectively to amino acids 36–50 and 194–201, which can participate in the GST–Jun–JNK association and inhibition, were tested.
Peptides corresponding to each of these domains were synthesized, and their ability to alter GSTp inhibitor activity was determined in vitro. As shown, the GSTp‐derived peptide from amino acids 194–201 (at the C‐terminal domain designated P1) abrogated GSTp inhibition of JNK activity (Figure 3F). Conversely, neither P2 (aa 36–50; Figure 3F) nor three unrelated, non‐GST‐based peptides of varying lengths (8–24 amino acids; not shown) were capable of affecting GSTp inhibition of JNK. Treatment of cells with TER‐199 (the prodrug form of TER‐117 which is converted to active form in vivo and has been shown to elicit effective inhibition of GST activity in vivo; Flatgaard et al., 1993) led to a 2‐fold increase in basal JNK activity in non‐stressed cells. UV irradiation also increased (∼2.5‐fold) JNK activation in TER‐199‐treated cells (Figure 3G). That TER‐199 also affected JNK activities after UV irradiation suggests that the dose of UV irradiation was insufficient to mediate complete inactivation of GST as a JNK inhibitor; thus, the presence of a specific GSTp inhibitor led to an additive effect on overall JNK activities in these cells. The effect of these GSH peptidomimetic agents is likely to be mediated either by altered conformation of the GSTp molecule or by a competition for the binding site, as non‐active (bacterially produced) GST was also capable of eliciting JNK inhibition (Figure 3B).
Changes in ROS affect GST oligomerization and its association with, and inhibition of, JNK
We next assessed whether modulation of the cellular redox potential would affect JNK inhibition by GSTp. We monitored the GST–JNK complex in vivo by means of immunoprecipitations followed by immunoblot analysis. Exposure of mouse fibroblast cells to either UV or H2O2 reduced the amount of the JNK–GSTp complex and increased JNK activity, whereas pre‐treating cells with the free radical scavengers N‐acetylcysteine (NAC) or the ethyl‐ester of glutathione (eeGSH) prevented JNK dissociation from GSTp and maintained GSTp inhibitory activity (Figure 4A I–III). These observations indicate that GST–JNK–Jun association is inversely correlated with JNK activity. To elucidate further changes to GST that take place upon altered redox conditions, we measured the possible formation of GST–GST dimers/multimers through disulfide bonds on neighboring cysteines. Monitoring the migration of GSTp on non‐reducing SDS–PAGE revealed decreased monomer/dimer forms of GSTp in UV‐ or H2O2‐treated cells (Figure 4A IV). Instead, a high molecular weight band corresponding to a multimer form of GSTp was detected (Figure 4A IV, upper arrow in panel a). Pre‐treatment with NAC or eeGSH maintained the lower molecular weight forms of GSTp (Figure 4A IV, the single arrow in panel a points to the dimer and panel b shows the monomer). H2O2 treatment is known to cause the formation of intra‐ or intersubunit disulfide bonds between cysteines at amino acids 47 and 101 of GST (Shen et al., 1993), resulting in a multimerization of GST subunits which no longer accommodates JNK molecules.
Monomer form of GSTp mediates JNK inhibition
The observation of changes in GSTp from monomer/dimer to multimer forms upon exposure to increased ROS prompted us to determine which of the two prevalent GSTp forms, monomer or dimer, exerts inhibitory activity on JNK. A gel filtration column was used to separate the monomer from the dimer form of GSTp (Figure 4B). A purified form of either monomer or dimer was added to a solid‐phase kinase reaction in which pre‐formed hisJunfull‐length–JNK complex served as a substrate. As shown, only the monomeric form of GSTp was capable of mediating efficient inhibition of JNK phosphorylation of c‐Jun (Figure 4B).
GSTp expression is inversely correlated with JNK activities
In a complementary approach to study GSTp effects on JNK activity, increasing amounts of GSTp cDNA were transiently transfected into mouse fibroblasts. As shown in Figure 5A, GSTp transfection resulted in a dose‐dependent decrease in JNK activation by UV. While transfection of 0.5 μg of GSTp caused a 20% decrease, 2 μg led to a 40% and 10 μg to a 50% decrease in JNK activation by UV irradiation. These observations suggest that it is possible to alter the degree of JNK activation by GSTp transfection, albeit within the relatively narrow range of 0.5–2 μg.
Effect of GSTp on phosphorylation of JNK kinase MKK4/JNKK/SEK1
To elucidate further the mechanism underlying GSTp inhibition of JNK activity, we monitored possible changes at the level of MKK4 phosphorylation. In vitro phosphorylation of JNK by MKK4 was not inhibited by GSTp (not shown). In non‐stressed 3T3 fibroblasts, there is a basal level of MKK4 phosphorylation, detected by MKK4 phospho‐antibodies (Figure 5BI). UV irradiation efficiently increased MKK4 phosphorylation. Conversely, forced expression of GSTp reduced the level of MKK4 phosphorylation in a dose‐dependent manner (Figure 5BI). Under the same conditions, GSTp did not alter phosphorylation of ERK1 (Figure 5BII). Treatment of GSTp‐transfected cells with TER‐199, an inhibitor of GSTp, restored the level of MKK4 phosphorylation (Figure 5BI). The observation that under physiological levels of GSTp expression MKK4 was phosphorylated, although JNK activity was inhibited by GSTp, suggests that GSTp does not affect JNK kinase under non‐stressed conditions. GSTp inhibition of JNK could be attributed to the nature of the complex between the two proteins. The finding that overexpression of GSTp reduces MKK4 phosphorylation suggests that an excess of GSTp can also affect the MKK4–JNK module.
Further evaluation of the possible interplay between GSTp and upstream JNK kinases was carried out in cells that had been transfected with ΔMEKK1, an MKK4 upstream kinase. Forced expression of ΔMEKK1 led to constitutive phosphorylation of MKK4, even when GSTp was co‐transfected. Interestingly, although GSTp was not able to reduce MKK4 phosphorylation in the presence of ΔMEKK, it efficiently blocked JNK‐mediated phosphorylation of c‐Jun (Figure 5BIII). These findings point to a selective effect of GSTp on JNK. GSTp inhibition of MKK4 depends on the level of GSTp expression and the signal elicited by the upstream kinase (as shown here for MEKK1). GSTp inhibition of JNK is due primarily to their association, which is released upon the conversion of GSTp from a monomer to a dimer form.
GSTp expression results in higher ubiquitination of c‐Jun
Since JNK efficiently targets the ubiquitination of its non‐phosphorylated associated proteins c‐Jun, ATF2 and p53, we determined the possible effects of GSTp on ubiquitination of JNK substrates in this reaction. Under non‐stress growth conditions, c‐Jun exhibits a short half‐life, which is prolonged upon phosphorylation by JNK (Fuchs et al., 1996, 1997; Musti et al., 1997). Transfection of GSTp cDNA into 3T3 mouse fibroblasts increased the level of c‐Jun ubiquitination in vivo (Figure 5C). Since the level of ubiquitinated Jun is inversely correlated with its degree of phosphorylation (Fuchs et al., 1996; Musti et al., 1997), the increase in ubiquitinated c‐Jun is an expected result of the GSTp inhibition of basal JNK activity, which reduces the number of c‐Jun molecules that undergo phosphorylation. The noticeable increase in ubiquitinated c‐Jun molecules provides an example of the physiological significance of JNK inhibition under normal growth conditions.
JNK activity in cells of GSTp null mice
We established embryo fibroblast cells from GSTP1/ P2(−/−) mice [MEFGSTP1/P2(−/−)] in order to assess JNK activity in a GSTp‐free environment. These cells do not express GSTp as monitored by either RT–PCR (data not shown) or Western blot analysis (Figure 6A). Stress in the form of UV irradiation, sorbitol or anisomycin markedly increased JNK activity in both MEFwt and MEFGSTP1/P2(−/−) cells; of these treatments, UV elicited the greater degree of JNK activation (Figure 6B). Interestingly, a higher basal level of JNK activity was found in the MEFGSTP1/P2(−/−) than in the MEFGSTwt (Figure 6B and C). This high activity could be diminished in a dose‐dependent manner upon transfection of GSTp cDNA (Figure 6C). Lesser amounts of GSTp cDNA were required to mediate 80–100% inhibition of UV‐mediated JNK activation in the MEFGSTP1/P2(−/−) cells, as compared with the MEFGSTwt cells (not shown). In both types of MEFs, transfection of GSTp caused 80–100% inhibition of UV‐mediated JNK activation, whereas in mouse fibroblast 3T3 cells, GSTp inhibition reached only 50%. Such differences may be attributed to the overall amount and form (monomer and dimer) of GSTp and/or the levels of other radical scavenger enzymes expressed in each of the cell lines.
To elucidate further the possible mechanism by which GSTp elicits its inhibition of JNK, we monitored levels of JNK phosphorylation before and after stress using JNK phospho‐antibodies. In MEFGSTP1/P2(−/−) cells, a higher basal level of JNK phosphorylation was seen (Figure 6D I), when compared with the MEFGSTwt cells. Forced expression of GSTp in these cells revealed an efficient reduction in the number of phospho groups on JNK, prior to, as well as after, UV irradiation (Figure 6D II). Forced expression of a truncated MEKK1 form (ΔMEKK1), which elicits constitutively high levels of JNK activity, partially restored the levels of JNK phosphorylation (Figure 6D II versus I). These observations are in line with the effect of GSTp on phosphorylation of MKK4, and suggest that GSTp elicits inhibition of JNK in vivo in spite of JNK phosphorylation by upstream kinases.
Forced expression of GSTp decreases transactivation of c‐Jun in GST null cells
MEFs derived from GSTp null mice [MEFGSTP1/P2(−/−)] as well as from GSTp wild‐type mice were co‐transfected with GSTp and Jun promoter (5× jun2 target sequence)‐driven luciferase constructs. MEFGSTP1/P2(−/−) cells exhibited high basal levels of transcriptional activity mediated by the Jun2 promoter sequence (Figure 6E, control transfection with empty construct, pcDNA3). This activity reflected the high basal JNK activity found in these cells (Figure 6B and C). Forced expression of GSTp efficiently reduced high basal Jun‐driven transcriptional activity in the MEFGSTP1/P2(−/−) cells (Figure 6E), implicating the capacity of GST to reduce JNK phosphorylation and activity (Figures 6C and D). As the amino acid residues which are important for GST–GST dimerization and enzymatic activity have been identified previously (Shen et al., 1993), we mutated GSTp at the respective sites and evaluated their ability to elicit JNK inhibition. When tested in MEFGSTP1/P2(−/−) cells, GSTp whose cysteines were mutated at amino acids 47 and 101 (which are required for GSTp dimerization) and GST whose tyrosine was mutated at amino acid 7 (which abrogates catalytic proton transfer activity) were as potent as wild‐type GSTp in inhibiting high basal JNK activity (not shown) and Jun‐mediated transactivation (Figure 6E). The lower basal levels of Jun‐mediated transactivation found in MEFGSTwt were also reduced by each of these constructs. The capacity of GSTp to reduce transcriptional activity mediated by the Jun promoter could be due to reduced phosphorylation of Jun and/or ATF2 by JNK, increased ubiquitination and degradation of c‐Jun, or both. These observations further support the hypothesis that GSTp inhibition of JNK does not require GST enzymatic activity and is mediated by its monomeric form. Forced expression of peptide derived from the GSTp C‐terminal domain (which blocked GSTp inhibition in vitro) increased basal JNK kinase and c‐Jun transcriptional activities, respectively (Z.Yin, V.Adler and Z.Ronai, unpublished observations). By monitoring c‐Jun transcriptional activities, these experiments point to the biological significance of GSTp as an inhibitor of JNK.
In the present study, we identify and characterize GSTp as a JNK inhibitor. Our data point to an additional cellular mechanism that is involved in the regulation of JNK activity before and after stress. In non‐stressed cells, the basal levels of JNK phosphorylation are low, in spite of constitutive stimulation by growth factors and endogenously formed ROS. Maintaining a low basal JNK activity is believed to affect the half‐life of JNK substrates, including c‐Jun, ATF2 and p53 (Fuchs et al., 1996, 1998c; Musti et al., 1997), and to play a key role in maintenance of controlled cell growth. Indeed, forced expression of GSTp in 3T3 fibroblasts increased the degree of Jun ubiquitination and decreased Jun‐mediated transactivation.
GSTp inhibition of JNK is found primarily in normally growing non‐stressed cells. Stress, as shown in the case of UV irradiation, decreases this inhibition in a dose‐dependent manner. Important to our understanding of GSTp's ability to elicit association with JNK and inhibition of this kinase is the finding that the inhibitory activities are confined to the monomeric form of GSTp. When first identified, the Jun–JNK‐associated protein had a mol. wt of 23 kDa, the characteristic size for the monomeric form of GSTp. Immunoprecipitation reactions always identified the monomeric form of GSTp as the Jun–JNK‐associated protein. Similarly, the ability of the monomer (but not the dimer) to elicit JNK inhibitory activity in vitro supports the role of the monomer GSTp in JNK inhibition in vivo. UV irradiation reduces GSTp–JNK association, probably as a result of the formation of GST–GST dimers and multimers; because of disulfide bond‐induced steric constraints, dimers/multimers cannot accommodate the Jun–JNK complex. ROS scavengers, such as NAC or eeGSH, inhibit the formation of GSTp multimers, prevent GSTp dissociation from Jun–JNK and maintain the low basal activity of JNK as a kinase. The switch from a monomer to a dimer/multimer form is likely to provide the underlying mechanism for GSTp's ability to sense and transmit changes in redox potential as a regulator of JNK signaling. Thus, based on its cellular conformation, GSTp dictates the association and inhibition of JNK. Importantly, while our studies demonstrate the effect of ROS elicited by UV and H2O2 on GSTp dimerization, certain type of stress (i.e. certain cytokines) are not expected to cause the same degree of ROS, and yet are strong inducers of JNK. The latter suggests that dissociation of GSTp from JNK may also occur due to changes other than disulfide bond‐based dimerization. The effect of post‐translational modifications of GSTp on its association with JNK is being investigated.
The finding that GSTp transfection reduces basal and UV‐inducible levels of JNK phosphorylation in vivo provides further insight into the mechanism by which GSTp elicits inhibition of JNK signaling. It is possible to rescue GSTp‐mediated inhibition of JNK phosphorylation by transfecting a constitutively active form of MEKK1. Independently, under physiological conditions, GSTp inhibition of JNK takes place in spite of activated MKK4, possibly through direct inhibition of JNK‐mediated Jun phosphorylation, supporting our original in vitro observation where GSTp inhibited c‐Jun phosphorylation by an activated form of JNK. The ability of GSTp to block JNK activity in spite of MKK4 and JNK phosphorylation by MEKK1 suggests that the nature of the GST–JNK association disables JNK activity as a kinase. That endogenous GSTp does not affect the MKK4 phosphorylation level in non‐stressed cells provides further evidence for selective inhibition of the Jun–JNK module by physiological levels of GSTp. That high expression of GSTp also reduces MKK4 phosphorylation could be attributed to possible inclusion of MKK4 with the GST–JNK module. Our data suggest that the interplay between upstream MKK4 signaling and the amount of monomer GSTp may determine the effect of GSTp on MKK4. Interplay between MEKK1–MKK4 and MKK4–JNK modules (Xia et al., 1998) and their existence in complex with scaffold protein, which determines the specificity of these signaling cascades (Whitmarsh et al., 1998), may explain the nature of changes elicited with different expression levels of GSTp. The specificity of GSTp‐mediated JNK inhibition was demonstrated via comparison of different protein kinases in a solid‐phase reaction, as well as in vivo, as forced expression of GSTp did not affect ERK1/2 phosphorylation. Our data also imply that MEKK1 is not a target for GSTp inhibition; other MKK4 upstream kinase(s), which include ASK1, TAKs, MLKs and GCKs, are currently being examined. Of interest is the observation that UV doses (10 J/m2) which decrease GST inhibitory activities are insufficient for JNK activation, which requires doses >20 J/m2. This suggests that conversion of the monomer to the dimer may precede JNK ability to elicit kinase activity MKK4/7, and is in agreement with the finding that GSTp efficiently reduces JNK phosphorylation of c‐Jun in vivo, even when its upstream kinase MKK4 is active.
That specific inhibitors of GSTp efficiently decrease GSTp inhibition of JNK both in vitro (TER‐117, TER‐287) and in vivo (TER‐199) suggests that their binding to GSTp is within the domain which is also required for association with Jun–JNK or, alternatively, that they alter the conformation of GSTp, affecting its association with JNK. Our results suggest that GSTp does not require transferase activity to mediate inhibition of JNK, in as much as bacterially produced GST, as well as GSTp mutated on Tyr7, which is essential for its catalytic activity, efficiently inhibited JNK enzyme and Jun transcriptional activities, respectively.
Important confirmation of the finding that GSTp is a regulator of JNK kinase activity comes from the use of cells from GSTP1/P2(−/−)‐null mice (Henderson et al., 1998). GSTP1/P2(−/−)‐derived MEFs revealed a noticeably higher basal level of JNK activity, which was also reflected in JNK's phosphorylation and Jun‐driven transcriptional activities. Transfection of GSTp into MEFGSTP1/P2(−/−) cells caused a decrease in high basal JNK phosphorylation, kinase activity and Jun‐mediated transactivation, further supporting the role of GSTp as an inhibitor of JNK signaling.
It is well documented that GSTp levels vary between different cell types (Tew, 1994). This, together with variable cellular compartmentalization and the expression of other detoxification enzymes, will contribute to variability of GSTp inhibition of JNK activity. Indeed, comparison of 3T3 fibroblasts and MEFs revealed that while GSTp transfection causes 100% inhibition in MEFs, it is limited to 50% in the 3T3 fibroblasts.
Overexpression of GSTp has been associated with transformation to malignancy (Sato, 1989) and acquired resistance to electrophilic anticancer drugs (Nakagawa et al., 1990; Tew, 1994). The finding that GSTp is a modulator of JNK inhibitor and the relationship between expression of this protein and JNK inhibition suggests that cancer cells prone to overexpress GSTp may exhibit high intrinsic JNK inhibitory activity. In this model, tumor cells overexpressing GSTp may escape apoptosis, which has been implicated as one of the end‐points of JNK activity (Xia et al., 1995; Kasibhatla et al., 1998).
The emerging model suggests that through its association with the Jun–JNK complex under non‐stressed conditions, GSTp inhibits JNK phosphorylation and activity (Figure 7). Changes in the level of ROS elicited by UV irradiation or H2O2 treatments decrease the amount of monomeric GSTp, resulting in a reduction of the GST–JNK complex, thereby enabling JNK phosphorylation (Figure 7). The fact that transcriptionally active p53 and c‐Jun directly affect transcription of radical scavenging enzymes including GST (Polyak et al., 1997; Komarova et al., 1998) points to the possible existence of an autoregulatory loop for GSTp regulation of JNK. Under such autoregulation, stress‐mediated GSTp dimerization/multimerization could enable JNK activation, yielding transcriptionally active p53/Jun and, in turn, newly synthesized GSTp that re‐forms a complex with JNK to limit the degree and duration of JNK kinase activity. Our studies support this model, which provides a possible underlying mechanism for the regulation of stress kinases by altered redox potential (Adler et al., 1996; Gomez del Arco, 1996; Kuo et al., 1996; Wilhelm et al., 1997; reviewed by Finkel, 1998). Thus, as a guardian of JNK activities in normally growing cells, GSTp may serve as a sensor of intracellular changes in redox potential that are elicited by various forms of stress.
Materials and methods
Cells and protein preparation
The mouse fibroblast cell line 3T3‐4A (Adler et al., 1995a), kindly provided by Dr Claudio Basilico, was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum and antibiotics (Gibco‐BRL). Cells were grown at 37°C with 5% CO2. MEFs from GSTP1/P2(−/−) cells and wild‐type control animals were prepared using standard protocols. MEFs were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) for no longer than 2 weeks. Proteins were prepared from cells as previously described (Adler et al., 1995a). In all cases, buffers contained a protease 'cocktail' (1 μg/ml of pepstatin, leupeptin and aprotinin) and phosphatase inhibitors (1 mM sodium orthovanadate and 5 mM sodium fluoride).
H2O2, eeGSH, GST and NAC were purchased fom Sigma.
Antibodies, immunoprecipitations and immunoblots
Antibodies to c‐Jun, PKA and ERK1 were purchased from Santa Cruz. Antibodies to JNK were obtained from PharMingen. Antibodies to CKII were purchased from Upstate Biotechnology and antibodies to phospho‐JNK were purchased from Promega. Antibodies to GSTs were as decribed previously (Ramgamathan and Tew, 1991; Henderson et al., 1998). Antibodies to the phosphorylated form of MKK4, ERK1/2 were obtained from New England Biolabs.
Immunoprecipitations were carried out using 1 mg of protein extracts and 500 ng of the antibodies, for 16 h at 4°C. Protein A/G beads (Gibco‐BRL) were added (15 μl) for 30 min at room temperature before washes were carried out in PBS supplemented with Tween‐100 (0.5%). Immunoblot analysis was performed using 50 μg of whole‐cell extract that had been separated on SDS–PAGE (10%) followed by electrotransfer to PVDF membrane. Ponceau staining was carried out to confirm equal loading, followed by blocking (5% non‐fat milk) and reaction with the appropriate antibodies (diluted 1:3000) for 16 h at 4°C. Reactions were visualized using enhanced chemiluminesence (ECL) reagents (Amersham). Analysis of GSTp migration was performed as indicated (Shen et al., 1993), using material that had been immunoprecipitated (0.3 μg of GSTp antibodies and 1 mg of proteins) from cells. Where indicated, separation under non‐reducing conditions was performed in SDS–PAGE without including the reducing agent β‐mercaptoethanol in sample buffer.
Peptides and inhibitor
Selective inhibitors of GST, including TER‐117, TER‐199, TER‐291 and TER‐317, were synthesized, purified to >93% purity, and kindly provided by Telik Inc. (San Francisco, CA). Peptides corresponding to flexible domains on GST [amino acids 194–201, sequence SSPEHVNR (P1), and amino acids 36–50, sequence TIDTWMQGLLKPTCL (P2)] were synthesized (Peptide Technologies Corp., Gaithersburg, MD) and purified by HPLC to >98.5%.
UV irradiation and H2O2 treatment
Cells in the logarithmic growth phase were exposed to UV‐C (60 J/m2, which requires a 15 s exposure in PBS with the lids off), followed by addition of medium and incubation for 45 min. When indicated, NAC (10 mM) was added to the cells 1 h prior to UV irradiation. For H2O2 treatment, medium from the culture dish was mixed with freshly diluted H2O2 (10 μM) and immediately applied to the fibroblasts (107 cells). When indicated, H2O2 was added in the presence of 1 mM eeGSH (Sigma).
Protein kinase assays
Protein kinase assays were carried out using a fusion protein, GST–Jun (amino acids 5–89; Adler et al., 1992) or hisc‐Jun (full‐length; Treier et al., 1994) as a substrate. JNK2 was purified from UV‐treated 3T3‐4A cells as previously described (Adler et al., 1995a). The purity of bacterially produced c‐Jun and of 3T3‐4A‐derived JNK was confirmed by silver‐stained SDS–PAGE. Solid‐phase JNK assays were carried out as previously described (Adler et al., 1995b, 1996). Briefly, the GST–Jun5–89 or hisJunfull‐length fusion proteins (0.5 μg/assay) were bound to glutathione–Sepharose or nickel beads, before incubation with the purified form of JNK (0.5 μg/assay) in the presence of kinase buffer [20 mM HEPES, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol (DDT), 2 mM MgCl2, 2 mM MnCl2, 5 mM NaF, 1 mM NaVO3, 50 mM NaCl] at room temperature for 15 min. The beads were pelleted and washed extensively with PBST [150 mM NaCl, 16 mM sodium phosphate, pH 7.5, 1% Triton X‐100, 2 mM EDTA, 0.1% β‐mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM benzamidine], before they were incubated with [γ‐32P]ATP (50 c.p.m./fmol; Amersham) in the presence of kinase buffer. Following extensive washing, the phosphorylated Jun was boiled in SDS sample buffer and the eluted proteins were run on a 15% SDS–polyacrylamide gel. The gel was dried, and phosphorylation of the Jun substrate was determined by autoradiography, followed by quantification with a phosphoimager (Bio‐Rad).
To assay for the presence of the JNK inhibitor, we used Jun–JNK as a pre‐formed complex. GST–Jun or hisJun (0.5 μg) was first pre‐incubated with the purified form of active JNK (0.5 μg), thus forming a Jun–JNK complex which subsequently was incubated with protein extracts (for 30 min) before addition of [γ‐32P]ATP (for 10 min) at room temperature. Important for the formation of these pre‐formed complexes is the ratio between Jun and JNK molecules (1:1 molar ratio) and the saturation of the nickel or glutathione beads with EDTA or glutathione to prevent non‐specific binding of cellular proteins. In all cases, the buffers used contained a cocktail of protease and phosphatase inhibitors (Adler et al., 1995c, 1997).
Other kinase assays were performed with peptides that are known to serve as specific substrates for Src tyrosine kinases, MAPKs and for PKA, all of which were purchased from Santa Cruz Biotech. Co. (Santa Cruz, CA). Histone H1 (Sigma) was used as a substrate for CKII. In all cases, the substrates were covalently bound to beads using the immunolink kit (Pierce) according to the manufacturer's recommendations. In these assays, 20 μl of bead‐coupled peptides (1 μg) were incubated with the source of kinase in the presence or absence of the inhibitor fraction, followed by extensive washing and quantification.
Purification of JNK inhibitor
Protein extracts prepared from 3T3‐4A mouse fibroblast cells (4 mg/ml) were precipitated with ammonium sulfate (50%). The precipitated material was dialyzed against kinase buffer and subjected to fractionation on small concentrators with cut‐off membranes of 100 and 30 kDa (Millipore, Bedford, MA). Material that passed through the 100 kDa membrane was placed on a 30 kDa column and the filtrate (<30 kDa) was concentrated further to a volume of 50 μl using a 10 kDa membrane (Amicon). Concentrated material that was 10–30 kDa in size was separated on a Superdex 75 column (SMART system; Pharmacia) pre‐calibrated with respect to the position of the expected molecular weight using a combination of protein standards. Using kinase buffer and a flow rate of 40 μl/min, fractions (200 μl) were collected and tested for inhibitor activity. In these assays, 20 μl fractions were added to pre‐formed hisJun–JNK complex before addition of [γ‐32P]ATP in the presence of kinase buffer and a cocktail of protease and phosphatase inhibitors. Active fractions were pooled and applied to a MonoQ column with a gradient of 20–500 mM NaCl in kinase buffer at a flow rate of 75 μl/min. MonoQ fractions were tested for inhibitory activity, and positive fractions were pooled and loaded on a phenyl‐Sepharose column in a buffer consisting of 20 mM K2HPO4 pH 7.5, 10% glycerol and 0.6 M ammonium sulfate. Using a gradient elution scheme, the inhibitory component was found in the 0.2 M ammonium sulfate fraction. To adjust for kinase buffer and to concentrate the fraction that contained inhibitory activity, the phenyl‐Sepharose eluate was concentrated on a 3 kDa column and applied to a pre‐formed hisJun–JNK complex. This fraction inhibited JNK activity (Table I). Material that was bound to the hisJun–JNK complex revealed a single protein on silver‐stained SDS–PAGE.
A large‐scale preparation of the JNK inhibitor (Table I) was subjected to multiple separation steps as described above, followed by SDS–PAGE and blotting onto a PVDF membrane. The purity of the single protein identified by Ponceau staining on the PVDF membrane was confirmed in parallel by silver staining of the same material. The band identified on the PVDF membrane was excised and subjected to N‐terminal analysis on an ABI protein sequencer Model 494 equipped with a Model 140C phenylthiohydantoin microanalyzer. The sequence Pro‐Pro‐Tyr‐Thr‐Val‐Val‐Tyr‐Phe‐Pro‐Val‐Arg‐Gly, which was obtained at the 10 pmol level, has 100% homology to the human GST P1‐1.
Purification of GSTp
GSTp was purified from human placenta. GSTα and GSTμ were purified from human liver as described (Ciaccio et al., 1991), followed by Superdex 75 gel filtration (SMART). Purity was confirmed by silver‐stained SDS–PAGE.
Transcription and ubiquitination assays
Transcriptional analysis of Jun was carried out using the 5× Jun2‐driven luciferase construct as previously reported (van Dam et al., 1998). In all cases, values were normalized with respect to transfection efficiency. In vivo ubiquitination assays were carried out by transfection of Jun and HA‐tagged ubiquitin into the 3T3 cells as indicated elsewhere (Treier et al., 1994).
We are grateful to Andras Schaffer, Mark Bluth, Zheng Dong and Theresa Wood for technical assistance, and to Alla Polotskaya and Serge Khilko for advice. We also thank Claudio Basilico for the 3T3‐4A cells, Dirk Bohmann for the hisJun and Ub‐HA constructs, Craig Monell of PharMingen for the antibodies to JNK, Michael Comb of New England Biolabs for the phosphoantibodies to MKK4 and ERK1/2, and Telik Inc. (San Francisco, CA) for the supply of GSTp inhibitors. The work was supported by NCI grant CA77389 to Z.R.
- Copyright © 1999 European Molecular Biology Organization


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THANK YOU to the cast and crew of the FABULOUS comedie musicale, Mozart l'Opéra Rock. It's second season started in early November of 2010 and tomorrow will be my 10th time seeing the show since it opened in '09. I am so happy that I could attend so many performances. It's truly my favourite show EVER!!!I will definitely miss going to see the show as it begins it's tour around France in February. The show will be back in Paris on July 8 and 9th only at Bercy so if you HAVEN'T yet bought your tickets, I recommend you do so.... NOW!!! It's worth every pretty penny!
I've also become friends with a number of other 'fans' and it's nice to have MOZART in common!
MOR - as some of us call it, has become a raging success in France as well as a few other French speaking countries. The producers of the show have just announced that the show will be translated into English and will become international! That's fantastic news and if it opens in London one day, you can BET that I'm hoping on the Eurostar to see it over there!
Thank you again to everyone involved in the show - on the scene and behind the scene- and even ABOVE the scene. And thanks to Dove Attia and Albert Cohen for such a TOP RATE and OUTSTANDING PRODUCTION!


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This weekly event is hosted by Breaking The Spine where bloggers spotlight upcoming releases that they are eagerly waiting patiently for.
Book that I am most eagerly anticipating this week is:
Publisher: Del Rel (Random House)
Release Date: Janurary 13th 2015
Genre: Sci-fi, Dystopia, Young Adult, Fantasy
Link: Goodreads
Synopsis (from Goodreads):
With shades of The Hunger Games, Ender's Game, and Game of Thrones, debut author Pierce Brown's genre-defying epic Red Rising hit the ground running and wasted no time becoming a sensation. Golden Son continues the stunning saga of Darrow, a rebel forged by tragedy, battling to lead his oppressed people to freedom from the overlords of a brutal elitist future built on lies. Now fully embedded among the Gold ruling class, Darrow continues his work to bring down Society from within. A life-or-death tale of vengeance with an unforgettable hero at its heart, Golden Son guarantees Pierce Brown's continuing status as one of fiction's most exciting new voices.


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The colour challenge: Blackberry Bliss, Hello Honey, And Tangelo Twist
After thinking about it for a while, I chose to use the stamp set "Wondrous Wreath"
- The finished card size is 4 1/2 by 4 3/4", with a 1/2" flap on the left side that folds over a piece of DSP.
- Here are the measurements:
Hello Honey 5 1/2 by 4 1/4" Scored at 4 3/4"
Whisper White: 4 1/4 by 3 5/8" (for sentiment)
Blackberry Bliss 3 7/8 by 3 5/8"
Blackberry Bliss DSP 5/8 by 3 5/8"
Whisper White 3 1/2 by 3 1/2" for wreath and bow
Dazzling diamond glimmer paper 4 by 2" - coloured with a Tangelo Twist marker
Jute ribbon 5"
- The wreath was made first. It was stamped first with Hello Honey, then with Tangelo Twist, and the berries with Blackberry Twist. The wondrous wreath framelit was used to die cut the wreath. The bow was die cut in Whisper White, and later coloured with a Tangelo Twist marker.
- The Jute ribbon was attached to the 35/8 by 3 7/8" Blackberry Bliss piece
- The strip of DSP was then adhered to the left side of the Hello Honey piece, and the Blackberry piece was adhered to the top of the Hello Honey.
- The wreath was adhered to the front of the card using dimensionals. I made glitter using dazzling details and a tiny drop of Tangelo Twist reinker, and applied it with a blender pen.
- "Noel" was die cut with Dazzling diamond glimmer paper and coloured with a Tangelo Twist marker. The Fine tip glue pen was used to adhere it to the front of the wreath.
- A rhinestone was added to the bow.
- The inside of the card was stamped with the sentiment from the stamp set.
Stamp: Wondrous Wreath
Inks: Hello Honey, Tangello Twist, Blackberry Twist, Tangelo Twist Marker
Papers: Blackberry Twist, Hello Honey, Whisper White, Blackberry Bliss DSP, Dazzling diamond glimmer paper
Accessories: Blender Pen, Dazzling Details, Tangelo Twist reinker, Fine Tip Glue Pen, Wondrous Wreath framelits, rhinestones


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I went back through my posts to determine what I'd already written about the Ironwork Quilt-Along quilt I started in 2012, and it turns out not much. Not sure how that happened, except around the time I started that quilt, Norm had a work accident and my attention was diverted elsewhere. Best guess as to why it fell through the cracks.
Anyway, all's well that ends well. Norm recovered in a few months, and almost two years later, I finally finished the Ironwork quilt!
The flimsy was done in 2012, probably not too long after I started it. Then it was put on the rack to properly age. In other words, I hadn't a clue how to quilt it, so there it sat. Fast forward to now and I finally felt ready to tackle the finish. It seemed to me that it needed a lot of ditch quilting around the rust-brown parts first, so I started there.
For quilting inspiration, I was thinking about what might naturally occur around an iron gate, and ivy came to mind. My heart-shaped leaves don't exactly look like ivy, but close enough for rock and roll, as they say.
Plan A had actually been a more controlled, larger, leaf-shaped design, done sort of on the order of feathers, i.e., around a central stem. However, that didn't pan out in practice. Hence Plan B was adopted, and meandering heart-shaped leaves it was.
I used a variegated Sulky top thread and Aurifil in the bobbin. Fabrics are Quilter's Candy Solids from Connecting Threads.
You can find the QAL posts for the Ironwork quilt at Piecemeal Quilts, HERE.
Thanks, Sandi, for the inspiration, beautiful quilt pattern, and great instructions. I really enjoyed making this!
Linking to:


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The World's best health care supplier is France. France spends about $4000 per particular person per 12 months on health care. France, with a population of sixty four million is spending more than US$250 billions of presidency expenditure on the health care of its people. Many varieties of health care interventions are delivered outdoors of health services. They embody many interventions of public health interest, similar to meals safety surveillance, distribution of condoms and needle-alternate programmes for the prevention of transmissible ailments. Serving to dental and orthodontics professionals improve their patients' oral health. You'll be able to put your personal stamp of uniqueness on a program by way of the promotions and incentives you employ.
What is going on on in your head? Till you recognize, there will be all sorts of problems. Uncorrectable problems. You possibly can't repair a problem that you don't see, proper? That is where mindfulness is available in. Mindfulness and mental health are intimately related. Your thoughts is busy in there proper now, talking about all kinds of things. It might be telling you that you are tired of your job, or that you are a victim of circumstance. It could be working via a listing of all of the issues it's worthwhile to be doing. It might be saying all of the fallacious things, and yet you may hear none of it. Chances are you'll just really feel a slight nervousness constructing as your day goes on.
According to some health experts, sugar not solely makes you achieve fats but may cause despair and lack of concentration too and that the common individual is three.5 stone heavier now than they have been back within the sixties because of increases in sugar consumption. It destroys collagen in the pores and skin that means you age … Read More ...


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Loooove me some pesto.
1/2 c grated Parm or pecorino cheese
3-4 cloves garlic
1/4 c roasted nuts – sunflower or pumpkin for the budget friendly version.
2.5 c lightly packed basil leaves (+ parsley if you want to dilute a bit)
1/2 t salt
1 T lemon juice (especially if you're freezing)
3/4 – 1 c EVOO (or grape seed)
Directions: if you're starting with block cheese, granulate this in the food processor with the blade attachment first and set aside. No need for the grater as the cheese is so hard.
Put whole, peeled cloves of garlic into the empty food processor and once minced keep them there. Add seeds and as much of the leaves as you can with a bit of oil and mix it up until combined. Add the rest of the leaves, salt and lemon juice and combine well. Stream in the oil and you're done!
I like to freeze this in ice cube trays and use it whenever I want. Who has all this time on a regular basis?!
Good reference for mixing it up http://www.bonappetit.com/test-kitchen/how-to/article/pesto-out-of-anything
If you have lots you can 4x or 6x this recipe and it just fits into one 13 quart food processor!


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Not The Tenhead!
I might have to go watch the trampoline foxes a million more times after hitting "publish" on this post, because this shit is making my forehead weep. A source who saw the photos of Alien Princess RiRi after she got a midnight beatdown from Chris Brown (allegedly) says her injuries are "horrific." The source told TMZ that she has two huge and swelled up "contusions" on both sides of her forehead. RiRi's lip is split, her nose is all sorts of bloody and she has bites marks on her fingers and on one arm. And Chris has got some fucking teefs on him! The dumb cunt could bite through a lead pipe covered in Kryptonite.
This is far from a "smack bitch once to shut them up" incident. This sounds like Chris got the evil spirit of Ike Turner in him and went to beat down town.
RiRi wasn't the one who called the police. A person who heard her screaming is the one who dialed 911. When the police arrived, Chris had already abandoned RiRi and the car by running off, but he took the keys with him. RiRi told the police Chris used his fists and there was no other kind of weapon involved. She refused treatment at the scene.
E! reports that RiRi is working with the fuzz, but isn't really telling them what caused Chris to get all Mike Tyson on her.
RiRi was supposed to perform in Malaysia this weekend, but of course, she has put that shit on hold and will reschedule at a later date. Chris Brown also canceled some shit he was supposed to do this weekend. Instead, he will spend his days enjoying his ride on the "You Fucked Yourself" express. Go on and take a bow, Chris. It's over now.
And seriously, out of all the things he beat down on, he had to go for her glorious tenhead! That's the portal to her home planet! SOS! That has to add another few decades to his sentence if found guilty. That is beyond illegal.


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The guy asked me, "I am sure you follow a spiritual discipline? Right?" I replied, "Yes... We dress modestly and our uniform reflects what we stand for and God's love. The uniform consists of five things which are signs of our love for God and it reminds us of our commitment and duty to God and humanity. We wake up early in the morning before sunrise. This can be from early as 1am to 4am or so. A Sikh meditates on the Divine-Name of God for at least one or two hours and then does morning prayers as a source of energy, inspiration and divinity. This recharges the battery of a Sikh... To see God in all, requires one to see the Divine within themselves first. How would you do this? This is through meditation on the Divine-Name of God. The morning meditation and prayer helps us to remember God and feel God's presence throughout the day." The guy replied, "Wow, this is amazing. I feel lazy now. I just remember God when ever I can. To be honest sometimes that is just when I wake up whilst lying in bed." I added, "When coming back from work, we recite an evening prayer to thank God for the day and get ourselves back in 'sync' and spiritually charged and then a prayer before sleeping so that we can sleep peacefully."
Later, the air hostess came over with drinks and snacks. I kindly declined to having a drink and snack. The guy asked, "Do you not eat out?" I replied, "I only eat food that has been prepared by spiritually disciplined people. I try to praise and remember God whilst preparing food so that food is not just food but spiritually satisfying food. Food cooked whilst meditating and thinking of God not just nourishes the body but provides stability to the mind and nourishes the inner-spirit.... The food you eat and how it is prepared affects our thoughts and mind. Nowadays children eat more fast food and as a result of eating fast food their minds have become 'fast'. Children who rely on fast food struggle to concentrate and pay attention. If one was to make food with love and positive thoughts then the eater will feel this. Compare eating a meal made by grandmother and a microwave ready meal." The guy looked amazed and said, "This is just amazing. Whatever you are saying seems to make sense and is 100% the truth."
The guy then asked me, "I sometimes feel low and down. I easily get affected by work and get run down by the world around me. What can I do?" I replied, "This is where spiritual discipline (Rehit) comes in... Life is not consistent and therefore you need a base to provide consistency and balance in this diversity. The one thing that we can try and become consistent in is our spiritual discipline. As a Sikh, our daily prayers, waking in the morning, the uniform we wear, the appearance the Guru has given us, the discipline of diet, the ordained lifestyle and expected behaviour helps to give consistency, inner-balance, support and acts as a shock absorber... This is why you have to have discipline, uniform, rules and regulations in school. The day children are given freedom to wear what they want, it leads to chaos and unruly behaviour. School children need consistency and rules to support them. I know this from previous experience as a school teacher." The guy listened very attentively and said, "You are 100% speaking the truth. You are so right. What you've said makes sense."
Lastly the guy asked me, "Can you please tell me how I can overcome my fears in life and stop being fearful." I replied, "My Guru tells me that to be fearless, you have to meditate on the Fearless One - God... Whoever you idolize, worship and remember you will become like them." With the short answer he became satisfied and said "Thank you."
The flight came to end and the guy shook my hand and said "Thank you for talking to me. You speak the truth and I am happy with things you told me. I am glad I sat next to a Sikh." I replied, "I have just shared with you Guru Nanak's teachings and wisdom. Happy to meet you as well."
Dhan Hai Guru, Dhan Hai Teree Sikhee!


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Bonding with our families should not be an option to make. It's always a responsibility to give time to each member of our family. Friends and 'barkadas' can come and go, but a family would never leave us no matter what would happen. I know that most of us are more comfortable being with friends, but that should not be the case. In life, only a family would never leave us alone.
|Amlan Bluewater Beach Resort


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Israel's Finance Minister: Financial Assistance to Settlements Grew Significantly During My Term
Yuval Steinitz says ministry had to compensate for construction freeze and 'discrimination' against settlements due to international pressure.
Israel's Finance Minister Yuval Steinitz said in an interview with religious daily Makor Rishon that during his term, assistance to settlement in the West Bank and in the Golan rose significantly.
Steinitz said that the assistance came because of his deep feeling "that in recent years settlements were discriminated against due to considerations stemming from international pressure and the fact that certain organizations were not investing in settlements in the territories out of fear that such a step would damage their fund-raising ability."
In a special interview to be published Friday by the newspaper, Steinitz said that the Israeli public does not appreciate enough the fact that it has jobs, while the U.S. and some European country are plagued by unemployment. "During the crisis, the middle class's living standards rose and poverty went down," he said. "But the media is distorting what is happening in reality."
Steinitz said that "we had to compensate the settlements for the damage done by the (construction) freeze and the slower pace of building – I am very proud of this." He added that he also "had the privilege to contribute much to the Ariel cultural center which was stuck for years because of budgetary problems, and to the Kiryat Arba cultural center." According to Steinitz, the ministry is now assisting the construction of a cultural center in Ma'aleh Adumin.
When asked about the evacuation of a house in Hebron earlier on Wednesday, Steinitz said that he supports these who settle the land of Israel and criticized the security establishment. "If Jews bought a house legally and with their own money, it is unclear why the security establishment is calling this a provocation," he said, adding that "any honest person should appreciate the courage and personal sacrifice of Jews who are making a brave Zionist move buying apartments and houses and settling in the heart of East Jerusalem and the Old City. This is very important in order to prevent the division of the city."
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Sheepily delicious! This looks like exactly the right addition to my spinning library at exactly the right time. Congrats on hatching the book, Beth. 🙂
Woo-hoo a spinning giveaway! Not just any spinning giveaway either – friend, breed enthusiast and expert spinner, Beth Smith has been working on this book for years and it's finally here!
This book picks up right where Deb Robson's Fleece and Fiber Sourcebook left off. It answers the question, sheep breed by sheep breed, "how do I work with it?" This book covers choosing, washing, prepping and spinning fleece. It's thorough and pretty amazing, just look:
Storey Publishing has donated a copy of this beautiful book for a giveaway.
Want to explore some of the breeds Beth Smith discusses in The Spinner's Book of Fleece? Enter Alison from The Spinning Loft. She built a raw fleece sampler around Beth and her book, The Beth Smith Sampler.
Here's her description: This sampler pack is a fantastic companion. Containing 2 oz. each of 15 breeds that Beth identifies and/or discusses in the book, this is a great way to get a taste for some of Beth's favorite fibers. Contents of the sampler vary based on stock, but each sample is found in the book.
How many people are so fiber obsessive they have a fleece sampler named after them? Alison has donated one sampler for our giveaway.
Ready to spin? Ready to win?
Our regular rules apply:
Sheepily delicious! This looks like exactly the right addition to my spinning library at exactly the right time. Congrats on hatching the book, Beth. 🙂
Can't wait to see this book, not sure I can hold off until a book signing. Bet the sampler is the perfect accompaniment – what a great giveaway!
OK, this is the giveaway I most want to win. Really.
Golly Gee, I hope it's me!
You were absolutely right when you said this is "not just any spinning giveaway" – that's a fabulous prize package! What a great way to learn more about fleece from various breeds, both in theory and practice!
It looks amazing! I'm really excited for this book.
I love the idea of having 15 breeds of fiber right there on hand to mess around with as I am reading the book! Chances are, I haven't worked with many of them, though I love breed specific spinning challenges.
That looks like an. Amazing book! What a guide to spinning different breeds, and with the sampler pack this is an awesome prize 😉
Would love to win! Great book!
JKnits51 on Ravelry
I so look forward to getting my hands on this book!
I spin. I knit. Book. Fleece. Best way to end the summer.
What an amazing resource!
What a wonderful giveaway! I would love to win. Thanks for the chance!
What an absolutely awesome giveaway!
Please include me in the list of hopeful people! What a prize!
Loved the Fleece and Fiber Handbook; this sounds like a really wonderful next step – I would love love love to win it!
wow! what a great prize and lovely addition to my library…
I'd love to win this prize!!
Congratulations Beth!
Wonderful giveaway. Would cherish.
Pretty Please pick me?
Bouncing up and down with excitement! So excited about this book!
What a beautiful prize. Storey Publishing and Alison are quite generous.
I am just learning to spin and this would be an amazing resource for me! 🙂
Wowza, what a great giveaway!
Thanks for a wonderful giveaway – I'm still a beginner but want to learn it all!
Would love to win this! If not, sigh, I'm seeing this on my Christmas list.
What a lovely looking book!
Omg! I need this! 😀
Looks so lovely. What a great giveaway.
Wow! What an awesome giveaway. I'm looking to get into spinning, so this would be super helpful!
Whilst I would love to win this, I'm in the UK and guess that the giveaway is US only? Still, this is the second time that I have come across this book in two days, and it has rave reviews. I think that I might add this to my Christmas list.
I am returning to spinning after a year long hiatus. I have a new wheel to renovate; and my old (now repaired) wheel, plus a little fluff on the spindle. I can't wait to get going again.
Neat giveaway! As an avid knitter, I wish I knew more about spinning.
What a lovely giveaway. That sampler to accompany the book is a stroke of genius!
What an awesome giveaway!
What a great book!
Love the idea of the sample pack.
This book sounds pretty awesome!
What a great giveaway! Thank you!
This kit is wonderful! Like a 'box of chocolate.' The book & sampler pack gives the spinner a tasting of lovely fiber. Just like a tasting party it looks like it shows you how to best enjoy it.
I would love to win this! Thank you for the opportunity
What a lovely giveaway, thank you!
Motivation to practice.
Looks like a beautiful book that I will have to have one way or the other!
This is such a great idea for a giveaway! I've been waiting for this book to come out for a while. The sampler pack is brilliant!
What a wonderful giveaway! Please add me to your "hopefuls" list, as this book and sampler would be an amazing addition to my spinning resources ;). Thank you for offering this contest.
Perfect way to get into the zone of…sample, sample sample!
I can't wait to get this book! Good luck for all of us!


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The week of teacher staff development has flown by and most teachers, including myself, still don't feel ready for Monday! In less than 48 hours, teachers will be on duty at my school ready to greet and begin teaching all of those eager (and not so eager) students!
Sunday night many teachers will lay in bed, tossing and turning! They will wake up every hour to check the time and make sure that they have not slept through their alarm!
Minds whirling all night cause sleep deprived teachers to stagger toward the coffee pot each morning hoping for that caffeine boost to get them through the day. It's almost like having a newborn at the start of each school year!
I found this pin about a bedtime yoga routine and decided to try it! I have to admit, I've felt more relaxed and have fallen asleep quickly (and stayed asleep) each night this week during our week of staff development.
The true test will come on Sunday night! Will I be able to sleep the whole night through or will I check that clock every hour on the hour?
I'll give you an update Monday.
(If I'm not too tired from lack of sleep)
The above image is from a great article I found giving advice to avoid teacher burnout! Here's the link!
Tips for the Yoga Link: Print it out so you don't have to keep scrolling while your trying to relax! Move the Savasana pose to the next to the last pose instead of 2nd.


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Friday, August 5, 2011
The fertility fears of the religious right
First, I must correct Ana. The USA is right at the replacement rate, and may actually be slightly under at 2.06. The population has grown largely due to immigration (which also includes all those South Korean, African, etc. babies people like to adopt, by the way). I added the above video to mainly focus on Steve King, particularly the part about a "dying civilization." At the end of the video, Sam suggests that this "part of a Sharia law...campaign." That is much of what it is. Part of it is likely also part of a pro-white campaign as well. The fertility rate of white people in this country is below the replacement rate. To better understand what King is saying, I present the following video:
This is the type of fear mongering that the right likes to spread. I think ideas like this are even worse in Europe, but we are starting to see more of it here in the USA as well. The video does a good job of mixing fact with fiction. It is true that the replacement rate is around 2.11. However, we start seeing fiction shortly after when the video says that a rate of 1.3 is "impossible to reverse," but then immediately goes on to say "because it would take 80-100 years to correct itself." Well, if it can correct itself, then it's not impossible to reverse, is it? "Impossible," then, is a buzz-word that attempts to exaggerate the seriousness of such a birthrate and feed fear. After that, we are told, "There is no economic model that can sustain a culture during that time." Well, they I suggest we get some economists on that and figure out a solution!
The video then goes over the birth rates across Europe. Some of those countries actually have higher birth rates. Spain is closer to 1.5, for example. However, I suspect they are looking at the non-Muslim population, since Spain has a high Muslim population. Because the next things they do is show how many Muslims are emigrating into Europe and look how they are breeding like rabbits!
Now, I grant I do have concerns myself. I do worry about the numbers of people who hold ridiculous religious beliefs possibly being on the rise, but I don't care about the religion. Fundamental Islam is just as much of a threat to humanity as fundamental Christianity. So, unlike these people on the religious right, the way to tackle this problem that I propose is to be critical of religious beliefs. In fact, let's be critical of any belief that is not founded on evidence. [Cringes at the fact I have not written a post on skepticism.] I have heard it said that the next generation of immigrants usually integrate into their society quite well. So, even if you have immigrants who are radical Muslims, their children could end up being quite different from their parents if the culture is not composed of radical Muslims. And that is why I think encouraging skeptical and critical thinking is the best solution. I don't give a crap if they are not white. People are people. Or I should say humans are humans. That, and skin color is skin deep.
I am also disappointed with the fertility rates of Muslims, like those on the right, but not because they are Muslims. Rather, I'm not happy that they are not helping to keep the world population down!
Also, where is the concern for eastern Asia? China - 1.54 fertility rate. (By the way, their economy has been doing OK thus far.) Japan - 1.21!!! If that doesn't send a strong message that this is all about fear mongering to stir up white Christians, I don't know what will.
At any rate, this is the sort of fear that drives the comments of those like Steve King. And it's the sort of fear that drove people like Anders Behring Breivik. The part that scares me is that this man does not appear to be the fundamentalist* Christian he was reported to be. He was sympathetic with Christian culture and knew some Bible verses, but he admittedly did not practice much. But what if he had actually been a fundamental Christian?
* It may be that he could be considered a fundamentalist Christian based on Norway's standards, as they are not a very religious nation. He would not have been a fundamentalist by American standards.
Posted by Leo Buzalsky at 1:55 PM
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Sunday, May 26, 2013
Captain Valiant-Marvin Stein-1945
3 comments:
You're right about the limited use of the hero, Captain Valiant. But by 1945, a lot of comics were already de-emphasizing the super-hero element. The Golden Age Daredevil appeared less & less in his own stories (taken over by the Little Wise Guys) & Blue Beetle often appeared in costume in only a few panels.ReplyDelete
I liked the artwork & sort of enjoyed those colored circle panels. They gave the story a little extra "pop".
Thanks for posting it. Fun.
Actually, the hero appears in costume in said fourteen panels, but out-of-costume in other panels — and in those out-of-costume panels that tendency to obscure his face continues. Indeed, look at the fourth panel of the third page, in which the (out-of-costume) character is reduced to a word balloon.ReplyDelete
The style looks like a cross between Howard Sherman and Fred Guardineer.ReplyDelete


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Date of sighting: April 1, 2012 7:37 AM
Location of sighting: Beijing, China
This was reported this morning in Asia. Also Know China time is a day ahead of USA. The person posted this from their cellphone directly.
Eyewitness states: "This UFO was in Beijing CBD Jingxian. Today I shot it in the morning from the CTS overpass. I was too interested in the traffic and the main road, when I pushed the shutter I actually had a UFO!"
[Click here to read full article]


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Blog about TV Series
Hi friends, have something in your minds: Comments, Critiques, Questions or Requests?. Just write them done. You know the rules : no spams, keep civilized, and polite.Every comment that against the rules above will be deleted.
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Here's a sample of what Momma's got for ya at the Flinders for you on the 6th Sept. I've whacked together a little bit of everything here, in fact this lil 70 minute mix has 19 trax covering 42 years from 1966 to 2008. We have some Northern Soul, 70's funk, 80's boogie, 90's uk soul, trip hop, break beats, NYC poetry, deep disco, nu electro funk, and fresh soul funk edits. I knocked it up in a hurry on the one n twos, please excuse it being a bit chunky in places (one take and half of it was blind selecting towards the end) but I think I've managed to make a bunch of relatively varied trax stack up together to give an insight to the vibes Momma wants to share with you. I hope ya dig it and I look forward to some feed back.
Aug 27, 2008
Track listing:
Bread & Butter
I should have known better
Bug powder dust
The real me
Feeling free
Unknown white label 7" (thanks Regal)
Late in the evening
Where the rainbow ends
Give it up
Bobby Whitney
Boogie no more
3 soaps in one
Nobody but you
Save me
I want your love
For those who like to groove
The magic man
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