text: News Releases << [Back to News Releases] Avista Corp. Reports Results for the Third Quarter of 2005 Q3 results reflect improvement for Avista Utilities and a net loss at Avista Energy primarily as a result of natural gas inventory SPOKANE, Wash. – October 27, 2005, 4:05 a.m. PDT: Avista Corp. (NYSE: AVA) today reported a net loss of $9.0 million, or $0.19 per diluted share, for the third quarter of 2005. For the third quarter of 2004, the net loss was $9.8 million, or $0.20 per diluted share. Avista Corp.'s net income was $19.8 million, or $0.40 per diluted share, for the nine months ended Sept. 30, 2005, an increase from $12.6 million, or $0.26 per diluted share, for the nine months ended September 30, 2004. "It continues to be a challenging year for Avista Energy. As was the case during the first quarter, substantial increases in natural gas prices have had a continuing negative effect on Avista Energy's earnings primarily due to the accounting treatment for Avista Energy's management of natural gas inventory. This negative impact is expected to reverse in future periods when the natural gas is withdrawn from storage,�? said Avista Chairman, President and Chief Executive Officer Gary G. Ely. "The utility results for the third quarter of 2005 were affected by higher electric resource costs and the absorption of excess costs under the Energy Recovery Mechanism dead band in the state of Washington. We believe that our utility operations are well positioned for a strong fourth quarter and modest improvement for 2006 primarily as a result of general rate increases. However, we are concerned about the effect that higher energy prices may have on our customers and ultimately our shareholders,�? Ely said. Results for the third quarter of 2005 and the nine months ended Sept. 30, 2005 (YTD), as compared to the respective periods of 2004: ($ in thousands, except per-share data) Q3 2005 Q3 2004 YTD 2005 YTD 2004 Operating Revenues $265,679 $241,552 $901,175 $811,172 Income from Operations $4,811 $4,545 $92,213 $84,339 Net Income (Loss) $(9,037) $(9,782) $19,756 $12,574 Business Segments: (contribution to earnings (loss) per diluted share) Avista Utilities $(0.04) $(0.15) $0.72 $0.26 Energy Marketing & Resource Management $(0.17) $(0.03) $(0.34) $0.08 Avista Advantage $0.03 $0.01 $0.06 --- Other $(0.01) $(0.03) $(0.04) $(0.07) SUBTOTAL (before cumulative effect of accounting change) $(0.19) $(0.20) $0.40 $0.27 Cumulative effect of accounting change – – – $(0.01) Total earnings (loss) per diluted share $(0.19) $(0.20) $0.40 $0.26 Recent Legal Developments On Oct. 19, 2005, the United States District Court for the Eastern District of Washington issued an order granting Avista's motion to dismiss a shareholder lawsuit. This complaint, originally filed against the company and certain current and former officers in September 2002, alleged violations of federal securities laws through alleged misstatements and omissions of material facts with respect to the company's energy trading practices in western power markets. The order of dismissal was issued by the Court without prejudice, allowing the plaintiffs 14 days to amend their complaint to address certain issues identified by the Court. Third Quarter and Year-to-Date 2005 Highlights Avista Utilities: Avista Utilities had a net loss of $1.8 million, or $0.04 per diluted share, for the third quarter of 2005, compared to a net loss of $7.3 million, or $0.15 per diluted share, for the third quarter of 2004. The third quarter is typically the lowest earnings quarter for Avista Utilities. The results for the third quarter of 2005 were further reduced primarily due to higher electric resource costs and the absorption of $8.2 million of the Energy Recovery Mechanism (ERM) dead band during the period, which included the reversal of the $0.7 million benefit from the first half of 2005. In 2004, the entire $9 million dead band was expensed during the first half of the year. The net loss for the third quarter of 2004 was primarily due to write-offs of $14.7 million ($9.6 million, net of taxes) related to the Idaho Public Utilities Commission (IPUC) general rate case order. Avista Utilities contributed $35.6 million to net income, or $0.72 per diluted share, for the nine months ended Sept. 30, 2005, an increase from $12.6 million, or $0.26 per diluted share, for the nine months ended Sept. 30, 2004. The increase reflects the positive effects of general rate increases implemented during the second half of 2004 in Washington and Idaho and the gain on the sale of Avista Utilities' South Lake Tahoe natural gas properties. Comparing 2005 to 2004, in 2004 year-to-date results were reduced by write-offs related to the IPUC general rate case order. On a year-to-date basis for 2005, Avista has expensed $7.5 million of the $9 million dead band and expects to absorb the remaining $1.5 million during the fourth quarter. The expensing of the $9 million dead band was anticipated in Avista's 2005 earnings guidance. During the period from January through the first half of March 2005, the Inland Northwest experienced an unusually warm and dry winter. However, during late March through the second quarter of 2005, precipitation and hydroelectric conditions improved. Avista expects hydroelectric generation will be approximately 94 percent of normal in 2005, even with a forecast of below normal precipitation and streamflows for the fourth quarter. This expectation is subject to change based upon precipitation, temperatures and other variables. The earnings impact of below normal hydroelectric generation is mitigated by regulatory mechanisms in Washington and Idaho that defer 90 percent of increased power supply costs for recovery in future periods, excluding the $9 million dead band in Washington. In March 2005, Avista Utilities filed a request with the Washington Utilities and Transportation Commission (WUTC) to increase its base electric and natural gas rates. On Aug. 12, 2005, Avista, the WUTC staff, the Northwest Industrial Gas Users and the Energy Project entered into a settlement that, if approved by the WUTC, would resolve all issues in Avista's electric and natural gas general rate cases. The Public Counsel Section of the Washington Attorney General's Office and the Industrial Customers of Northwest Utilities did not join in the settlement agreement. Hearings were held in October 2005, and the parties have requested that the settlement agreement become effective January 1, 2006. The WUTC can take several actions with respect to the settlement including, but not limited to, approving it as presented, rejecting it and ordering additional rate case proceedings, or approving it with conditions that would also be subject to the acceptance of the settling parties. The revised rate increase requests in the settlement are designed to increase annual electric and natural gas revenues by $22.1 million and $1.0 million, respectively. The majority of the increase in electric revenues provides for an increased level of power supply costs to be recovered in base rates. As such, this portion of the increase will not increase gross margin or net income. The settlement agreement provides for an overall rate of return of 9.11 percent, including a return on common equity of 10.4 percent based on an equity level of 40 percent. Under the settlement agreement, Avista has agreed to increase the utility equity component to 35 percent by the end of 2007 and 38 percent by the end of 2008. If these targets are not met, it could result in a reduction in base retail rates. The utility equity component was approximately 31 percent as of September 30, 2005. The settlement agreement also provides for modifications to the ERM, including a reduction in the dead band from $9.0 million to $3.0 million. In addition, the current ERM surcharge of 9.8 percent would be increased to 10.8 percent to increase the rate at which deferred power costs are recovered. During the third quarter of 2005, Avista filed purchased gas cost adjustment requests in Washington, Idaho and Oregon to increase natural gas rates to reflect higher natural gas costs. Oregon natural gas rates were increased 22.5 percent effective October 1, and Avista has received approval for a 23.5 percent increase in Washington to become effective November 1. Avista has requested a 23.8 percent increase in Idaho to become effective November 1. These natural gas rate increases are designed to pass through changes in purchased natural gas costs to customers with no change in Avista Utilities' gross margin or net income. The company filed its license application with the Federal Energy Regulatory Commission (FERC) in July 2005 for the Spokane River Project representing five hydroelectric plants on the Spokane River. Avista's current license for the Spokane River Project expires on Aug. 1, 2007. The company has requested the FERC to consider a license for Post Falls that is separate from the other four hydroelectric plants. This is due to the fact that Post Falls presents more complex issues that may take longer to resolve than those dealing with the other facilities of the Spokane River Project. Energy Marketing and Resource Management: This business segment had a net loss of $8.3 million, or $0.17 per diluted share, for the third quarter of 2005, compared to a net loss of $1.2 million, or $0.03 per diluted share, for the third quarter of 2004. The net loss for the third quarter of 2005 was primarily related to losses in Avista Energy's natural gas portfolio with a significant portion of the loss due to the accounting for the management of natural gas inventory that is further discussed below. The net loss for the third quarter of 2004 was due to an impairment charge related to a generating asset owned by Avista Power. This business segment had a net loss of $16.9 million, or $0.34 per diluted share, for the nine months ended Sept. 30, 2005, compared to net income of $3.8 million, or $0.08 per diluted share, for the nine months ended Sept. 30, 2004. The net loss for year-to-date 2005 was primarily related to losses in Avista Energy's natural gas portfolio during the first and third quarters. Increases in natural gas prices had an overall negative impact on results for 2005. While Avista Energy's portfolio is within its internally established limits and in accordance with its risk management practices, losses can and do occur when the market moves contrary to Avista Energy's positions, which has occurred during 2005. As markets moved counter to certain positions, Avista Energy acted to adjust its position consistent with established risk management practices. This reduced the market risk but had the effect of locking in losses that were recorded during the first and third quarters of 2005. These losses can be magnified when there are large unforeseen fluctuations or disruptions in the market, such as those caused by the hurricanes in the southeastern United States during the third quarter. Avista Energy continued to produce positive results on the electric side of its business that includes trading, marketing and managing the output and availability of combustion turbines and hydroelectric assets owned by other entities. Avista Energy continues to seek opportunities to expand its business of optimizing generation assets owned by other entities and has expanded its natural gas end-user business to commercial and industrial customers in Montana. The operations of Avista Energy are managed on an economic basis, reflecting all contracts and assets under management at estimated market value consistent with industry practices, which is different from the required accounting for certain contracts and physical assets under management. Under Statement of Financial Accounting Standards No. 133, "Accounting for Derivative Instruments and Hedging Activities,�? certain contracts that are considered derivatives and accounted for at market value, economically hedge other contracts and physical assets under management that are not considered derivatives and are generally accounted for at the lower of cost or market value. The accounting treatment does not impact the underlying cash flows or economics of these transactions. These differences are generally reversed in future periods as market values change or the contracts are settled or realized. These differences primarily relate to Avista Energy's management of natural gas inventory, as well as Avista Energy's control of natural gas-fired generation through a power purchase agreement. These differences had a $7.7 million negative effect on results for the third quarter of 2005 and accounted for almost 70 percent of the net loss for Avista Energy during the nine months ended Sept. 30, 2005. Avista Energy anticipates that losses of approximately $12 million, net of tax, associated with natural gas inventory will be reversed when the natural gas is withdrawn from storage, which is forecasted to occur during the first quarter of 2006. However, the loss could increase or decrease prior to that time depending on changes in natural gas prices. In July 2005, Avista Energy amended its committed credit agreement to increase the amount available from $110 million to $145 million and to extend the expiration date by two years to July 2007. Avista Advantage: This business segment completed its fifth consecutive quarter of positive earnings, contributing $1.3 million, or $0.03 per diluted share, for the third quarter of 2005. This is compared to net income of $0.4 million, or $0.01 per diluted share, for the third quarter of 2004. For the nine months ended Sept. 30, 2005, Avista Advantage contributed $3.0 million to net income, or $0.06 per diluted share, compared to net income of less than $0.1 million for the nine months ended Sept. 30, 2004. Through Sept. 30, 2005, Avista Advantage has already exceeded its original earnings guidance for the full year of 2005. The improvement for the third quarter and year-to-date 2005 over 2004 was primarily due to an increase in operating revenues from customer growth, as well as the settlement of an employment contract during 2004. Avista Advantage's revenues increased by 38 percent for the nine months ended Sept. 30, 2005, as compared to the same period one year ago, while the average cost of processing a bill decreased by 7 percent for the same period. Other Business Segment: This business segment had a net loss of $0.2 million, or $0.01 per diluted share, for the third quarter of 2005, compared to a net loss of $1.6 million, or $0.03 per diluted share, for the third quarter of 2004. For the nine months ended Sept. 30, 2005, this business segment had a net loss of $2.0 million, or $0.04 per diluted share, compared to a net loss of $3.4 million (excluding the cumulative effect of accounting change), or $0.07 per diluted share, for the nine months ended Sept. 30, 2004. Liquidity and Capital Resources: During the nine months ended Sept. 30, 2005, positive cash flows from operations, proceeds from the sale of Avista's South Lake Tahoe natural gas distribution properties and a reduction in the company's total cash position were used to fund the majority of Avista's cash requirements, including utility capital expenditures and dividends. Utility capital expenditures totaled approximately $150 million, the most significant of which was the acquisition of the remaining interest in Coyote Springs 2. For 2006, Avista is establishing a utility capital budget of approximately $160 million. In September 2005, Avista Corp. terminated the lease agreement with and acquired the Rathdrum CT from WP Funding LP (an entity whose financial statements have been consolidated since 2003). As a result of this transaction, Avista Corp. is no longer including WP Funding LP in its consolidated financial statements as of Sept. 30, 2005. This transaction and deconsolidation did not have a material effect on the company's total consolidated assets, liabilities, stockholders' equity or results of operations. From a consolidated perspective, the $56.3 million of WP Funding LP debt and third-party investment was replaced with borrowings on Avista Corp.'s committed line of credit. Avista Corp. expects to issue long-term debt during the fourth quarter of 2005 to, among other things, refinance these borrowings. The balance outstanding under Avista Corp.'s five-year $350 million committed line of credit increased $89 million during the nine months ended Sept. 30, 2005, to $157 million as of Sept. 30, 2005, primarily to fund the debt redemption and maturities including the WP Funding LP transaction. Excluding the planned long-term debt issuance during the fourth quarter of 2005, Avista expects cash flows from operations and Avista Corp.'s committed line of credit to provide adequate resources to pay dividends, fund capital expenditures, repay maturing long-term debt and meet other contractual commitments. Earnings Guidance and Outlook For 2005, Avista is revising its guidance for consolidated earnings to a range of $0.65 to $0.75 per diluted share from a range of $0.95 to $1.05 per diluted share, due to the underperformance of the Energy Marketing and Resource Management segment. The company still expects Avista Utilities to contribute in the range of $0.95 to $1.05 per diluted share for 2005. The outlook for the utility assumes normal weather and temperatures, and below normal hydroelectric generation for the fourth quarter of 2005. The 2005 outlook for the Energy Marketing and Resource Management segment has been revised to a range of a loss of $0.30 to $0.35 per diluted share from a range of a loss of $0.05 to a contribution of $0.05 per diluted share. The revised outlook for this segment assumes stable natural gas prices and no positive or negative effect of accounting for the management of natural gas inventory. For the year 2005, the company expects Avista Advantage to contribute approximately $0.08 per diluted share and the Other segment to lose $0.05 per diluted share. For 2006, Avista is initiating its guidance for consolidated earnings to be in the range of $1.30 to $1.45 per diluted share. The company expects Avista Utilities to contribute in the range of $1.00 to $1.15 per diluted share for 2006. The outlook for the utility assumes normal weather, temperatures and hydroelectric generation, as well as the implementation of the Washington general rate increase as designed in the settlement agreement on January 1, 2006. The 2006 outlook for the Energy Marketing and Resource Management segment is a range of a contribution of $0.20 to $0.30 per diluted share, excluding any positive or negative effects of changes in prices on the accounting for the management of natural gas inventory. The company expects Avista Advantage to contribute in a range of $0.10 to $0.12 per diluted share and the Other business segment to lose $0.05 per diluted share. In addition to the earnings forecasts for 2006, if natural gas prices remain at September 30, 2005 levels, the company estimates that an additional $0.25 of earnings per diluted share will be recognized in consolidated earnings in the Energy Marketing and Resource Management segment when natural gas is withdrawn from storage. This is forecasted to occur during the first quarter of 2006. Avista Corp. is an energy company involved in the production, transmission and distribution of energy as well as other energy-related businesses. Avista Utilities is a company operating division that provides service to 330,000 electric and 285,000 natural gas customers in three Western states. Avista's non-regulated subsidiaries include Avista Advantage and Avista Energy. Avista Corp.'s stock is traded under the ticker symbol "AVA.�? For more information about Avista, please visit www.avistacorp.com. Avista Corp. and the Avista Corp. logo are trademarks of Avista Corporation. NOTE: Avista Corp. will host a conference call on October 27, 2005, at 10:30 a.m. EDT to discuss this report with financial analysts. Investors, news media and other interested parties may listen to the simultaneous webcast of this conference call. To register for the webcast, please go to www.avistacorp.com. A replay of the conference call will be available from 12:30 p.m. EDT on October 27 through 11:59 p.m. EST November 3, 2005. Call (888) 286-8010, passcode 56776911 to listen to the replay. The webcast will be archived at www.avistacorp.com for one year. The attached income statement, balance sheet, and financial and operating highlights are integral parts of this earnings release. This news release contains forward-looking statements, including statements regarding the company's current expectations for future financial performance and cash flows, capital expenditures, the company's current plans or objectives for future operations, future hydroelectric generation projections, and other factors, which may affect the company in the future. Such statements are subject to a variety of risks, uncertainties and other factors, most of which are beyond the company's control and many of which could have significant impact on the company's operations, results of operations, financial condition or cash flows and could cause actual results to differ materially from the those anticipated in such statements. The following are among the important factors that could cause actual results to differ materially from the forward-looking statements: weather conditions, including the effect of precipitation and temperatures on the availability of hydroelectric resources and the effect of temperatures on customer demand; changes in the utility regulatory environment; the impact of regulatory decisions; the potential effects of any legislation or administrative rulemaking passed into law; the impact from the potential formation of a Regional Transmission Organization; the ability to relicense the Spokane River Project at a cost-effective level with reasonable terms and conditions; volatility and illiquidity in wholesale energy markets; changes in wholesale energy prices; changes in global energy markets; wholesale and retail competition; unplanned outages at any company-owned generating facilities; unanticipated delays or changes in construction costs; natural disasters that can disrupt energy delivery; blackouts or large disruptions of transmission systems; changes in industrial, commercial and residential growth and demographic patterns; the loss of significant customers and/or suppliers; failure to deliver on the part of any parties from which the company purchases and/or sells capacity or energy; changes in the creditworthiness of customers and energy trading counterparties; the company's ability to obtain financing; the impact of any potential change in the company's credit ratings; changes in future economic conditions in the company's service territory and the United States in general; changes in rapidly advancing technologies; the potential for future terrorist attacks; changes in tax rates and/or policies; changes in, and compliance with, environmental and endangered species laws, regulations, decisions and policies; the outcome of legal and regulatory proceedings concerning the company or affecting directly or indirectly its operations; employee issues, including changes in collective bargaining unit agreements, strikes, work stoppages or the loss of key executives, as well as the ability to recruit and retain employees; changes in actuarial assumptions and the return on assets with respect to the company's pension plan; increasing health care costs and the resulting effect on health insurance premiums; and increasing costs of insurance, changes in coverage terms and the ability to obtain insurance. For a further discussion of these factors and other important factors, please refer to the company's Annual Report on Form 10-K for the year ended Dec. 31, 2004 and Quarterly Report on Form 10-Q for the quarter ended June 30, 2005. The forward-looking statements contained in this news release speak only as of the date hereof. The company undertakes no obligation to update any forward-looking statement or statements to reflect events or circumstances that occur after the date on which such statement is made or to reflect the occurrence of unanticipated events. New factors emerge from time to time, and it is not possible for management to predict all of such factors, nor can it assess the impact of each such factor on the company's business or the extent to which any such factor, or combination of factors, may cause actual results to differ materially from those contained in any forward-looking statement. << [Back to News Releases]


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Remember that show "Queer Eye for the Straight Guy?" On one of the episodes Thom Filicia, the "design doctor" used apple green to decorate one of the straight guy's bathroom. I would've never thought to use that color in such a small space, but ever since that transformation I've loved the color! And the bathroom turned out great, by the way. So today's post is a nod to that vibrant color ... are you ready to decorate with apple green? A pretty, … [Read more...]
Are You Ready to Decorate with Apple Green?
May 6, 2017 By Jennifer 17 Comments
17 Comments Filed Under: home decor, interior design Tagged With: apple green, color, decorating

subset: commoncrawl


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It feels like a while since I wrote something for this blog.
You will probably have noticed I haven't been writing for my blog much lately nor have I done any more translations. The reason behind that is that I am very much preoccupied with my full time job these days. That said, I still play games and watch the odd Anime show… Just not as much as I used to.
For the very same reason it's been a while since I went out for a photo stroll and practice all the photography techniques I've been reading up about here and there.


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Charity trustee or school governor?(5 Posts)
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bob schieffer
Veteran CBS Anchor Bob Schieffer Retires After Decades As A Newsman
"The one thing I'll never forget is the trust you placed in me and how nice you were to have me as guest in your home over so many years," Schieffer told viewers Sunday during his final broadcast.
You Can Watch The Debate Like It's A Choose Your Own Adventure
A pretty neat video that makes the final presidential debate interactive. Watch what you want, skip the rest. Powered by Bedrocket-Interlude.
Should Men Be Allowed To Moderate Debates?
Two moderators provoked lively, interesting debates this cycle. Two did not.
Why Mitt Romney Kept Talking About Mali
The West African country — likely obscure to most watching the foreign policy debate — just entered the national conversation.
The Presidential Debate Drinking Game, Foreign Policy Edition
Make a very sober discussion about foreign policy very unsober! Obama and Romney square off in their third and final presidential debate. Let's make sure it's one to not remember! Pourin' policy debate!
Herman Cain's Double-Talk On "Face The Nation"
On Sunday's Face the Nation, Herman Cain defended his wacky campaign ad that has his chief of staff Mark Block smoking a cig. "Let Mark be Mark," Cain said. "Let people be people." That's when host Bob Schieffer nailed him for running an irresponsible ad, and the GOP candidate started singing a different tune. (via gtcha.me) Watch Video ›


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09 May 12
Health economics is one of those rapidly growing fields that links up business with health care. A health economist has a unique passion for using their knowledge of economics by applying it to a variety of challenges in health and medicine. As the population continues to grow, the demand for quality and cost-effective health care similarly grows, leaving careers in health economics vital to sustaining our health care system
01 May 12
Medimetrics, recently certified to ISO 13485, has successfully developed the IntelliCap system, specifically designed to target delivery of small molecule drugs and biologics to the GI tract. The technology is capable of achieving controlled release of drugs to specific GI sites in a fast, cost-effective and convenient manner (Shimizu et al., 2008).
Michael Crawford
10 Apr 12
Why is Facebook on its way to become a $100 billion dollar enterprise? Because, the corporate world is very comfortable with 'predictability driven marketing' surrounded by 'search based social mediums' expects 'click based' returns on investments and believes in 247/365 multidirectional expansion while rest of the world loves the freedom to engage online.
Naseem Javed
28 Mar 12
If you want to go further in your clinical research career, the Institute of Clinical Research (ICR) can help. We provide training, analysis and discussion of the issues facing clinical research today, and opportunities for widening your professional network. Join ICR today, and benefit from all these resources straight away, many of them at specially discounted rates.
28 Mar 12
Strontium ranelate (Protelos ®) reduced structural progression of osteoarthritis (OA) in knee joints by one third, finds an international phase III study. The trial, presented at the European Congress of Osteoporosis and Osteoarthritis (IOF-ECCEO) meeting in Bordeaux, France, 21 to 24 March, 2012, is the first study ever to have demonstrated a disease modifying effect in OA, with additional benefits shown for pain, function and mobility.
Janet Fricker
28 Mar 12
Here's what employers have to look forward to in the coming month hopefully along with some much needed sunny spring weather!
Jim Golby
21 Mar 12
Engineering and sales skills top the list of those in most demand, according to employer feedback canvassed for the 2012 Hays Salary Guide.
Kathy Lou, Marketing Manager of Hays in China
15 Mar 12
Research proves that green spaces are restorative and therapeutic; they provide a relief from stress, a place for peace, a place for exercise, and a place to create connections with other people in our community.
Kelly Caulfield, Marketing & Communications
14 Mar 12
Financial and budgetary, IT and green skills top the list of ten skills that Hays' global offices and clients commonly identify as being in greatest demand.
"Talent shortages are a global problem," said Simon Lance, Regional Director of Hays in China. "We operate in 31 countries and these skills are the ones that our clients globally say are in most demand. For anyone considering their career options in our globalised economy, these are the skills to focus on.
Kathy Lou, Marketing Manager of Hays in China
27 Feb 12
This year is another bumper year for employment law changes. In this article we consider the key changes for employers in 2012, planned changes to Employment Tribunal practice and procedure and what changes are still in the pipeline.
Boyes Turner
PharmiWeb.com is Europe's leading industry-sponsored portal for the Pharmaceutical sector, providing the latest jobs, news, features and events listings.
The information provided on PharmiWeb.com is designed to support, not replace, the relationship that exists between a patient/site visitor and his/her physician.


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Emotional Djokovic serves up warning to his rivals
Novak Djokovic is into the last eight with a shout that was heard far and wide across the All England Club.
Those in the immediate vicinity, most notably an unfortunate ball girl, certainly felt it, but the defending champion's tenacity against Kevin Anderson will also have been grimly noted by the other seven quarter-finalists.
The startling scream came in the fifth set of his victory and Djokovic himself admitted it spurred him on to triumph, even if it also forced the nearby ball girl to turn as white as the All England Club's regulation kit.
"It was a very intense fifth set," Djokovic said. "Because he was winning his service games so easily, it put an extra pressure on my service games.
"That was maybe one of the turning points, that game where I won that very close point.
"I just took out everything I had, not on anybody but me. I was looking at the box, but I was talking to myself."
It just so happened that the ball girl, towel at the ready, had strayed into the blast radius.
"I'm sorry," added the Serb, who prevailed 6-7, 6-7, 6-1, 6-4, 7-5. "There was nothing towards her. Maybe she was just afraid of my screaming there. I was pretty close to her."
By completing the fourth comeback of his career from two sets down, Djokovic extended his record of reaching 25 consecutive Grand Slam quarter-finals and booked a Centre Court showdown with Marin Cilic, the US Open champion, today. But how he was made to fight for it in a match he rated as one of the toughest of his career.
"Until the last moment, until the last point, I didn't know if I was going to win or no," he said.
Even after levelling the match at two sets all on Monday before bad light stopped play, Djokovic had to face a renewed avalanche of Anderson aces on No 1 Court yesterday.
The 6ft 8ins South African with a reach seemingly as high as Table Mountain sent down three 130mph aces in his opening service game of the fifth set to set an ominous tone.
The frustration mounted for Djokovic. Not only was Anderson serving exceptionally, but he was dominating most of the backcourt exchanges, forcing a couple of break points in the fourth game that the champion saved.
In the sixth game, Anderson sent a forehand fractionally long and Djokovic's emotions came to the surface.
Although not a practising Buddhist, Djokovic is a frequent visitor to the Buddhapadipa Temple close to the Wimbledon.
Such tranquillity and harmony are on occasions no substitute for the cathartic release of a good old fashioned roar.
"Emotions are present. Always going through ups and downs. I was trying to get myself motivated," he said.
"I was not showing much emotions yesterday. I was just trying to keep it together. Sometimes it's just good to scream and let it all out, because that's the way I work."
It is testimony to the consistent pressure that Anderson applied that Djokovic was so far removed from his comfort zone.
How cruel then that a serve that had produced 40 aces would eventually prove Anderson's downfall when a pair of double faults gave Djokovic the chance to break in the 11th game.
Djokovic closed out the game, which extended to 3 hours 47 minutes over the two days, but dismissed the idea that his physical and mental reserves have been diminished.
"It was a big challenge. Now that I managed to survive, it adds more confidence. I'm going to be fine for the quarter-final. I haven't spent too much energy this tournament."
Meanwhile, Andy Murray goes into his quarter-final against Vasek Pospisil determined to avoid the sort of mental meltdown that gripped him 12 months ago.
Last year, Murray went into his quarter-final against Grigor Dimitrov on an apparently faultless run of form, only to suffer an inexplicable power fade and lose in straight sets.
To go out in such limp fashion - as defending champion too - made it the most disappointing result of his Wimbledon career to date.
Perhaps today's quarter-final against world No 56 Pospisil will turn into another car crash, but it seems unlikely.
Where Murray was at his most turbulent against Dimitrov - wild-haired, foul-mouthed, yet still strangely heavy-footed - he has barely uttered a curse in 2015.
Perhaps marriage really has helped him find a steadier equilibrium. And we should probably credit the input of his coach, Amelie Mauresmo, as well.
Asked if she noticed greater serenity about Murray, Mauresmo replied: "I hope, I hope, but he's improved a lot in that area as well.
"He is feeling great in the team, he's feeling the people around him are really committed 100 per cent. He feels he is out there performing at his best, so that helps him to be more serene on the court."
Pospisil has his own distinctive approach to the mental side of the game. He takes a purple notebook on court, placing it on the chair next to him at the changeovers and consulting it from time to time - just as Pete Sampras once melted Wimbledon hearts by reading an inspirational love letter from his wife Bridgette Wilson.
(Not that it stopped him losing to Georg Bastl in a contender for the title of greatest Wimbledon upset in history.)
"I have been looking at the same page for the matches, but the book is getting filled up a little bit," Pospisil said after his five-set win over Viktor Troicki on Monday.
"It's just reminders and pointers, things I should be looking at during the match, so that will change a little bit match to match.
"I'm working on my mental side of the game," added Pospisil, who also explained that he worked on some "things" every day for half an hour before breakfast.
When asked about these "things", he said they were secret, but we can assume he is talking about visualisation and other psychological push-ups.
The investment seems to have paid off, for Pospisil is the only -unseeded player left in either draw. (© Daily Telegraph, London)
Live, BBC 1/Setanta Ireland,
from 11.30


text: HerosRogues_HarryLoisHiggins Almost love at first bite! Harry met Lois on a Philippine beach when he spotted the dorsal fin of a shark right behind a young woman in the water. Without delay he charged in and rescued her! They soon married and settled in Salmon Arm. Harry was principal at J.L. Jackson School for 17 years. Lois, raised 3 kids and helped found Shuswap Theatre, the Naturalist Club and now sits on the Shuswap Community Foundation. ~ In our 100th Year: Salmon Arm, BC


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This weeks Humane Society showcase adoption pet Blaine
From staff and wire reports - Apr 11, 2013
Blaine is a full grown five-year-old male border collie mix.
Humane Society officals said Sal is "sweet and does well with children and other pets. He is a people watcher and exceptionally bright! He just wants a home and family. His life in a kennel everyday is pulling him down. Please consider adopting him, you wont be sorry."
In addition to this weeks showcase pet, Humane Society officials said there are plenty of very playful pets whose only wish is to have a home.
The St. Charles Animal Parish Shelter is located at 921 Rue Cannes in Luling.
For more information on pet adoptions, call (985) 783-5010.
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GET Geosciences Environnement Toulouse
PubMed | GET Geosciences Environnement Toulouse, Tomsk State University, University of Santiago de Compostela, Albert Ludwigs University of Freiburg and University of Naples Federico II
Type: | Journal: Journal of colloid and interface science | Year: 2015
Terrestrial mosses are commonly used as bioindicators of atmospheric pollution. However, there is a lack of standardization of the biomonitoring preparation technique and the efficiency of metal adsorption by various moss species is poorly known. This is especially true for in vitro-cultivated moss clones, which are promising candidates for a standardized moss-bag technique. We studied the adsorption of copper and zinc on naturally grown Sphagnum peat moss in comparison with in vitro-cultivated Sphagnum palustre samples in order to provide their physico-chemical characterization and to test the possibility of using cloned peat mosses as bioindicators within the protocol of moss-bag technique. We demonstrate that in vitro-grown clones of S. palustre exhibit acid-base properties similar to those of naturally grown Sphagnum samples, whereas the zinc adsorption capacity of the clones is approx. twice higher than that of the samples from the field. At the same time, the field samples adsorbed 30-50% higher amount of Cu(2+) compared to that of the clones. This contrast may be related to fine differences in the bulk chemical composition, specific surface area, morphological features, type and abundance of binding sites at the cell surfaces and in the aqueous solution of natural and cloned Sphagnum. The clones exhibited much lower concentration of most metal pollutants in their tissues relative to the natural samples thus making the former better indicators of low metal loading. Overall, in vitro-produced clones of S. palustre can be considered as an adequate, environmentally benign substitution for protected natural Sphagnum sp. samples to be used in moss-bags for atmospheric monitoring.


text: Send Enquiry add to multibuy What is a Multi-Buy? Free delivery on this item Please call our sales team for delivery dates Trustpilot This superb built in electric double oven from Bosch features a 67 litre main oven and a 35 litre secondary oven. This model features an A/B energy rating and is finished in stunning stainless steel. Please be aware that this model will not fit under your worktop, it must be fitted into a tall housing unit. 2 yearwarranty A/B Warranty Options Get added peace of mind with an extended warranty. Select your extended warranty at checkout. Find out more... 5 years inclusive warranty - £69.00 Features Colour: STAINLESS STEELEnergy rating: A/B LOOKING FOR MULTIPLE BOSCH APPLIANCES? CALL OUR TEAM OF EXPERTS TODAY FOR SOUND ADVICE AND A GREAT PACKAGE DEAL!Pop out rotary dials and touch controls Electronic clock Main lower oven: Net capacity 71 litres2 cooking functions:3D hot air, defrost Fully automatic programmerControl panel lock Interior light Smooth enamelled oven interior Easy fit shelf support rails 5 shelf positions EcoClean® back liner Secondary top oven: Net capacity 35 litres2 cooking functions: Full width variable grill, conventional top and bottom heat Smooth enamelled oven interior Interior light Easy fit shelf support rails 2 shelf positions EcoClean® back and roof liners Both ovens:Heat reflective glass Full glass inner door Telescopic shelf rails available as optional accessory Required electricity supply 20A


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Lubbock Christian University marine biology program
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Find info concerning Lubbock Christian University marine biology program, and registering for elective seminars and lectures. Which college degrees give you the best chances of finding employment after graduation? Seek out accredited nursing programs for the best training. Given a brief hospital internship or prior work experience, you can expect a great return on your medical education. Concentrating on Lubbock Christian University marine biology program, for example, you may gain an advantage in job recruiting on-campus. Check with the marine biology department for course registration deadlines, and oceanography program requirements. Please use the form above to request admissions info for Lubbock Christian University.
Biology is one of the original natural sciences, even reaching back as far as the study of anatomy in ancient Egypt, where evidence has been found of surgery, forensics, and anatomical knowledge of both organ structure and function. Biology in general is the study of life and living organisms, with specific attention to function, growth, evolution, identification and taxonomy. Modern biology is becoming an interdisciplinary field, overlapping with biomedical engineering, molecular chemistry, and genetics. The biological sciences recognize the cell as the basic unit of life, and DNA as the basic unit of heredity. Moreover, all organisms survive by consuming and transforming energy via respiration or photosynthesis, and by regulating their internal environment via homeostasis.
The life sciences are defined by the scale at which organisms are studied, in that biochemistry focuses on chemical processes of life while molecular biology involves molecules and protein synthesis. Cellular biology may be broken down further into a structural analysis of cell organelles and lipid membrane boundries, such as in skin tissue or alveolar sacs in the lungs. In the field of genetic engineering, DNA (deoxyribonucleic acid) may be spliced together from different organisms, in an attempt to transfer hereditary traits that may increase crop yields, create a supply of stem-cells, or even clone sheep. Beyond major requirements within the marine biology department, you may consider Moraine Valley Community College marine biology major as a complementary elective to your primary course of study.
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Frank Hyldgaard
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Mobil: 30681820
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From August 2nd till 6th the roads of Herning Municipality will host the European Championships.
The best bikeriders in Europe will ride the highways and byways of Denmark, when they in august 2017 will decide who becomes European Champion.
On August 8nd 2017 UEC (Union Européenne de Cyclisme) revealed that the 2017 European Road Cycling Championships will be held in Herning, Denmark in cooperation with DCU (Denmarks Cycle Union), Herning Municipality, Sport Event Denmark, Region Central Jutland and the MCH-cooperation.
- DCU as well as Herning Municipality and Sport Event Denmark have convinced us in UEC that they can lift the 2017 championship to new heights, for both the athletes and the spectators. We are, amongst many others things, excited about the ideas to involve the audience and have recreational cyclists try out the official route. At the same time, Hernings experience with hosting big sport events has convinced us that they are the right venue for an event of this magnitude, says David Lappartient, president of UEC.
DCU-president Henrik Jess Jensen is thrilled.
- It is fantastic to announce a five-day championship in Herning on august 2nd to august 6th. It will be a gigantic cycling-party, where junior, U23 and elite-riders will compete for 12 sets of medals. I would like to thank UEC for this opportunity and Herning Municipality and Sport Event Denmark for making this ambition reality, says Henrik Jess Jensen.
In Herning Municipality mayor Lars Krarup looks forward to a grand experience for both athletes and spectators.
- Herning is bicycle-city number 1 in Denmark, as we also demonstrated when we hosted the first two stages of Giro d'Italia in 2012. A European Championship lasting five days, with more than 700 riders in six classes will mark a new highlight in the municipalitys history, says Lars Krarup
The championships have backing from a wide array of organisations, including Sport Event Denmark, who has an aim to attract international championships to Denmark.
- Yet another large-scale cycling-event has been secured for Denmark, and yet again in a sport, where Denmark has proud traditions and great results. Previously Denmark has hosted World Championships on the road, on track and in BMX, as well as the world championships for amateurs last year. Now it is time for a great experience for the people of Denmark and a spectacular event in central Jutland. It will be festive and innovative, says Lars Lundov, director of Sport Event Denmark
The MCH-cooperation will handle a large part of the logistics and hosting at the European Championships, and director of MCH Georg Sørensen is looking forward to welcome the cycling-elite.
- At MCH we love to have guests, and an European Championship in cycling will generate a lot of them. As part of this event we look forward to utilizing our know-how and experience, as well as showing all of Europe, how our facilities can serve as a festive and successful setting for a championship, says Georg Sørensen
To the hosts it is important that the championships will be an including event. It is the plan to have several recreational races, where everybody can ride the same route as the professionals.
- We see great perspectives in UEC's vision to make ERCC one of the most important dates on the race-calendar The championships in Bretagne in 2016, where Peter Sagan won, set the bar very high, but we plan to top that in 2017 says, Henrik Jess Jensen.
Mayor Lars Krarup expect to see many international tourists along the route.
- In august, when Germans, Swedes and Norwegians visit the Danish west-coast in large numbers we see a good potential for attracting an international crowd. In 2012, with the start of the Giro, we had many international guests, and in 2017 the timing is even better. A race featuring the biggest stars of European Cycling will draw large numbers, says Lars Krarup.
2017 European Road Cycling Championships takes place from august 2nd to august 6th 2017.
ERRC is arranged by UEC, DCU, Herning Municipality, Sport Event Denmark, Region Central Jutland and MCH.
In the coming months the routes will be planned in cooperation with local police authorities.
In 2016 in Bretagne 777 riders from 45 countries participated in the ERRC.
The EERC will feature 12 different competitions in TT and roadrace for men and women in the Junior, U23 and Elite-classes.
The 12 competitions are planned like this:
Wednesday, August 2nd:
TT - Junior (M+W)
Thursday August 3rd:
TT - Elite (W+M)
Friday August 4th:
RR - Junior (W) + Junior or U23 (M)
Saturday August 5th:
RR - Junior or U23 (M)
RR - U23 + Elite (W)
Sunday August 6th:
RR - Elite (M)
Henrik Jess Jensen, president Dansk Cykle Union, 2520 2370
Lars Krarup, mayor of Herning Municipality, 9628 2322
Lars Lundov, director Sport Event Denmark, 4031 4855
Georg Sørensen, director of MCH, 21 400 200
Enrico Della Casa, Secrétaire Général, UEC, 0039 342 803 34 87
Frank Hyldgaard, projektmanager Herning Municipality, 3068 1820
Frank Hyldgaard
Tlf.: 96288643
Mobil: 30681820
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Previous experiments suggest a connection between the N-alpha-acetylation of proteins and the sensitivity of cells to apoptotic signals. Here, we describe a novel biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the anti-apoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We conclude that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.
Increasing evidence suggest that specific metabolic alterations associated with cancer cells may not be ancillary to their transformation but instrumental to their tumorigenic potential by mediating cell proliferation, growth and survival (Vander Heiden et al., 2009). Many oncogenes and tumor suppressor genes known to promote excess cell proliferation also alter biosynthetic (or anabolic) processes. For example, Akt expression stimulates glucose uptake and glycolysis, the pentose phosphate pathway and fatty acid synthesis. c-Myc expression promotes glutamine metabolism as well as purine and pyrimidine biosynthesis. Furthermore, mutations in genes encoding metabolic enzymes have been identified by cancer genetic association studies (Vander Heiden et al., 2009). However, how specific metabolites contribute to increased proliferation and apoptotic resistance in tumor cells remains a central unanswered question.
The proto-oncogene Bcl-xL has a prominent role in promoting cell survival and cancer development (Boise et al., 1993). It is well-established that Bcl-xL protects against apoptosis by directly binding and inhibiting Bax/Bak-oligomerization mediated mitochondrial permeabilization. However, certain Bcl-xL mutants, such as F131V/D133A and G148E, that are unable to bind to Bax or Bak, nevertheless retain 70–80% anti-apoptotic activity of WT Bcl-xL (Cheng et al., 1996). Curiously, Bcl-xL has also been shown to regulate mitochondrial respiration and metabolism (Gottlieb et al., 2000; Vander Heiden et al., 1999). Whether the metabolic function of Bcl-xL contributes to its role in mediating apoptotic resistance is unclear.
Our unexpected identification of an N-terminal acetyltransferase, Arrest Defective 1 (dArd1) in a genome-wide RNAi screen in Drosophila cells for apoptotic regulators (Yi et al., 2007) prompted us to posit that protein N-alpha-acetylation, a major N-terminal modification, links cell metabolism to apoptotic induction in cancer cells. Since dARD1 is epistatic to Diap1, a direct inhibitor of caspases in Drosophila, and ARD1 is required for caspase activation in mammalian cells (Yi et al., 2007), the role for ARD1 in mediating caspase activation is evolutionarily conserved. How ARD1 regulates caspase activation has not yet been illustrated.
In mammalian cells, protein N-alpha-acetylation is mediated by the highly conserved N-acetyltransferase protein complexes (NatA, NatB, NatC, NatD, and NatE). The NatA complex consists of the catalytic subunit, Arrest Defective 1 (hNaa10p/ARD1), and the auxiliary subunit, N-acetyltransferase 1 (NAT1/hNaa15p/NATH); whereas NatB consists of N-terminal acetyltransferase 3 (hNaa20p/NAT3) and mitochondrial distribution and morphology 20 (hNaa25p/Mdm20). Although the Nat complexes are implicated in regulating cell cycle progression, cell proliferation and tumorigenesis, the mechanisms that connect N-alphaacetylation to the cellular protein apparatus are unknown (Ametzazurra et al., 2008; Polevoda and Sherman, 2003; Starheim et al., 2008; Starheim et al., 2009). Recent N-acetylome studies reveal incomplete acetylation status of proteins (Arnesen et al., 2008; Goetze et al., 2009). Although a commonly accepted view is that partial acetylation results from degenerate nature of protein N-terminal sequences, we considered the possibility that protein N-alpha-acetylation might be regulated, an alternative hypothesis that had not been tested due to technical limitations.
Here we developed a novel biochemical approach to assess the status of endogenous levels of protein N-alpha-acetylation. Using this assay, we show that protein N-alpha-acetylation levels are sensitive to alterations in metabolism and Bcl-xL expression. Bcl-xL overexpression leads to reduced levels of acetyl-CoA and hypoacetylation of protein N-termini through a Bax/Bak independent mechanism. Conversely, Bcl-xL−/− mouse embryonic fibroblasts show increased levels of acetyl-CoA as well as protein N-alpha-acetylation levels. Protein N-alpha-acetylation deficiency in Bcl-xL overexpressing cells contributes to apoptotic resistance since increasing acetyl-CoA production can rescue this deficiency in protein N-alpha-acetylation and sensitize Bcl-xL cells to cell death. Our study suggests that regulation of acetyl-CoA availability and protein N-alpha-acetylation may provide a Bax/Bak independent mechanism for Bcl-xL to regulate apoptotic sensitivity.
We confirmed that ARD1 is necessary for cell death induced by the DNA damaging agent, doxorubicin, in multiple cell lines of different origins including Drosophila Kc cells (Yi et al., 2007), HeLa, HT1080, and U2OS cells (Figure 1A–D). In addition, HeLa and U2OS cells deficient for NATH were also resistant to doxorubicin treatment, recapitulating the apoptotic resistant phenotype of ARD1 knockdown cells (Figure 1A–D). Thus, the acetylation activity of the NatA complex serves to influence the sensitivity of these cells to apoptosis. Next we tested whether NatA influences apoptotic sensitivity to other DNA damaging agents. We found that ARD1 knockdown cells are also resistant to cisplatin and UV treatment (Figure 1E). However, these cells remained sensitive to tumor necrosis factor (TNFalpha) and cyclohexamide treatment, which specifically activates apoptosis through the death receptor pathway (Figure 1F). Thus, we conclude that protein N-alpha-acetylation regulates apoptotic sensitivity downstream of DNA damage.
Since N-alpha-acetylation has been suggested to affect protein stability (Polevoda and Sherman, 2003), we examined whether protein synthesis and/or protein turnover might be affected by acetylation status. We tested whether ARD1 substrates such as caspase-2 and Chk1 (see results below) are destabilized in ARD1 knockdown cells using cyclohexamide, an inhibitor of protein synthesis. Deficiency in ARD1 did not lead to decreases in the cellular levels of these proteins compared to that of control (Figure S1A). The steady state levels of total cellular proteins in ARD1 knockdown cells were similar to the levels in control cells (Figure S1B). We also tested whether general protein stability is altered in ARD1 or NATH knockdown cells (Figure S1C). By pulse-chase 35S-Met labelling experiments, we observed that neither general protein synthesis nor turnover was affected in ARD1 or NATH knockdown cells. Thus, protein N-alpha-acetylation mediated by NatA complex is not required to maintain protein stability globally. In addition, we verified that cell cycle progression is unaffected in cells deficient for ARD1/NATH (Figure S1D). Taken together, these data suggest that the NatA complex may influence apoptotic sensitivity by mediating protein N-alpha-acetylation of key apoptotic components.
The lack of an immunological method to detect the acetylation status of protein N-termini has limited our understanding of the mechanisms that regulate protein N-alpha-acetylation. To this end, we developed a selective biotin labelling method using an engineered protein ligase, termed subtiligase (Abrahmsen et al., 1991; Tan et al., 2007) that detects non-acetylated N-termini of endogenous proteins. This approach was used to capture unmodified protein N-termini resulting from caspase mediated cleavage during apoptotic cell death (Mahrus et al., 2008). Unblocked N-termini can be labelled using subtiligase, which preferentially biotinylates N-terminal amine groups consistent with the specificity of NatA or NatB (Abrahmsen et al., 1991; Mahrus et al., 2008). As the N-termini of up to 80–90% of cellular proteins may be blocked by a number of different modifications (Martinez et al., 2008), very few proteins will be biotin labelled by subtiligase as previously demonstrated (Mahrus et al., 2008). Thus, any protein that is biotin labelled by subtiligase in our assays most likely results from a specific loss in N-alpha-acetylation.
We utilized subtiligase to biotinylate free N-termini of proteins in whole cell lysates followed by avidin affinity purification and western blot analysis. Decreased levels of protein N-alpha-acetylation are expected to increase subtiligase-mediated protein biotinylation and conversely, increased levels of protein N-alpha-acetylation are expected to decrease subtiligase-mediated protein biotinylation (Figure 2A). First, we asked whether the subtiligase assay could be used to distinguish the N-alpha-acetylation status of protein N-termini when the expression of the NatA complex is diminished by RNAi mediated knockdown. ARD1 acetylates a subclass of proteins with Ser, Ala, or Thr as the newly exposed N-terminal residue after initiator Met (iMet) cleavage (Polevoda and Sherman, 2003). We tested 14-3-3β, which is known to be N-alpha-acetylated (Arnesen et al., 2009; Martin et al., 1993), and proteins that we predict to be N-alpha-acetylated based on their sequences, Chk1 and Msh2. Caspase-2, which is responsive to both DNA damage (Tinel and Tschopp, 2004) and metabolic stress (Nutt et al., 2009; Nutt et al., 2005), is also a good candidate for acetylation by ARD1 as the second amino acid in the caspase-2 polypeptide is Ala. We observed that these proteins as well as caspase-2 were biotinylated to a higher extent by subtiligase in NATH or ARD1 knockdown cells than in control cells (Figure 2B and 2C). These data suggest that subtiligase can distinguish N-alpha-acetylation of multiple proteins that is dependent on NatA expression.
To determine the validity of subtiligase assay, we measured the extent of protein N-alpha-acetylation by quantitative mass spectrometry using differential isotope labelling (Figure 2D). First, we tested whether we could detect the basal levels of N-alpha-acetylation of caspase-2 by mass spectrometry. We observed that the mass to charge ratio (m/z) of the N-terminal peptide of caspase-2 is shifted as expected with an acetyl modification (Figure S2A and S2B). Furthermore, we found a 30% reduction in the amount of N-alpha-acetylated caspase-2 in NATH deficient cells relative to control by subtiligase assay as well as mass spectrometry (Figure 2B; Figure S2D). These results support the conclusion that caspase-2 is N-alpha-acetylated by ARD1.
As caspase-2 is a substrate of ARD1 (Figure 2 and S2) and the activation of caspase-2 is inhibited by ARD1 or NATH knockdown (Figure 3A), we asked how N-alpha-acetylation of caspase-2 might influence caspase activation.
First we conducted mutagenesis analysis of caspase-2 to disrupt protein N-alpha-acetylation. We replaced the third residue of caspase-2 with Pro (A3P) as the presence of Pro in this position inhibits protein N-alpha-acetylation. The 3P mutation has been previously demonstrated to inhibit N-alpha-acetylation of other substrates, known as the XPX rule (Goetze et al., 2009). We also replaced the second Ala for Ser as a control to maintain N-alpha-acetylation (A2S) as well as iMet removal (Arnesen et al., 2009; Goetze et al., 2009). Generation of these targeted substitutions allows us to definitively test whether subtiligase can differentiate between acetylated and unacetylated forms of caspase-2. An increase in subtiligase-mediated biotinylation of A3P was detected, while very little A2S or wild-type caspase-2 was detected after biotin pull down, consistent with acetylation as the explanation for the lower biotinylation levels (Figure 3B). A defect in N-alpha-acetylation of A3P caspase-2, but not WT and A2S caspase-2 was confirmed by mass spectrometry (Figure S2A–C). Thus, subtiligase is an effective tool for detecting unmodified protein N-termini.
The caspase-2 scaffolding complex, which promotes caspase-2 activation, includes the adaptor protein, receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD) (Duan and Dixit, 1997; Tinel and Tschopp, 2004). The ability of the N-terminal caspase-2 mutants to interact with RAIDD was assessed by co-immunoprecipitation. We found that RAIDD efficiently co-immunoprecipitated with WT and A2S but not with A3P caspase-2 (Figure 3C). This suggests that N-alpha-acetylation of caspase-2 facilitates its interaction with RAIDD.
Since acetyl-CoA is a key co-factor in N-alpha acetylation, we speculated that the levels of N-alpha-acetylated caspase-2 might be dependent on expression of key metabolic enzymes that are responsible for production of cytoplasmic acetyl-CoA. To explore this question, we tested whether knockdown of ATP citrate lyase or acetyl-CoA synthetase, the two metabolic enzymes that utilize citrate and acetate, respectively, to generate acetyl-CoA, results in decreased levels of N-alpha-acetylated caspase-2. Indeed, we observed increased biotin labelling of caspase-2 in knockdown cells compared to control cells following subtiligase assay (Figure 3D). This suggests that caspase-2 is hypoacetylated when acetyl-CoA generation is reduced and therefore, protein N-alpha-acetylation is subject to metabolic regulation.
Since decreased levels of protein N-alpha-acetylation leads to apoptotic deficiency, we reasoned that regulation of protein N-alpha-acetylation might provide a mechanism for certain apoptotic regulators to control apoptotic sensitivity. Bcl-xL, an anti-apoptotic Bcl-2 family member, is known to have an effect on metabolism (Shimizu et al., 1999; Vander Heiden et al., 2001). We asked whether protein N-alpha-acetylation levels are sensitive to Bcl-xL expression using subtiligase assay. An increase in biotin labelling of caspase-2, -9, -3, and Bax was observed by Bcl-xL expression in 293T, HeLa, and Jurkat cells compared to that of control (Figure 4A, 4B, and 4C). Conversely, a decrease in biotin labelling was apparent in Bcl-xL−/− MEFs compared to that of Bcl-xL+/+ MEFs (Figure 4D). Since Bcl-xL is known for maintaining mitochondrial integrity by blocking oligomerization of Bax/Bak, we measured the levels of protein N-alpha-acetylation in Bax/Bak deficient cells. Surprisingly, the levels of protein N-alpha-acetylation were similar in Bax−/−, Bak−/− or Bax/Bak−/− DKO MEFs compared to that of WT MEFs by subtiligase assay (Figure 4E). This suggests that Bcl-xL mediated regulation of protein N-alpha-acetylation is independent of Bax/Bak.
Recent studies show that histone lysine acetylation is dependent on acetyl-CoA production in yeast and mammalian cells (Takahashi et al., 2006; Wellen et al., 2009). However, we found that lysine acetylation of histone H3 and H4 were unaffected in Bcl-xL cells compared to control (Figure S3). This suggests that histone lysine acetylation is not sensitive to the changes in acetyl-CoA levels associated with Bcl-xL expression.
We next tested whether protein N-alpha-acetylation levels in Bcl-xL cells are affected by changes in acetyl-CoA metabolism. Addition of acetate or citrate stimulates cytosolic acetyl-CoA production by acetyl-CoA synthetase or ATP citrate lyase respectively (Sullivan et al., 1974a; Sullivan et al., 1974b). We verified that these metabolites increase acetyl-CoA levels in mammalian cells (data not shown). Under metabolite treatment, protein N-alpha-acetylation levels were restored in Bcl-xL expressing cells to that of control levels (Figure 4A and 4B). Thus, a reduction in acetyl-CoA production in Bcl-xL cells may be responsible for the observed hypoacetylation.
The expression of Bcl-xL is often elevated in tumors (Adams and Cory, 2007). To explore the effect of Bcl-xL on tumor metabolism, we conducted a systematic search using a combination of 2D-NMR and mass spectrometry to identify metabolic changes associated with increased Bcl-xL expression. Principal component analysis (PCA) of one-dimensional (1D) proton spectra shows that the metabolome of Bcl-xL expressing cells was significantly different from the metabolome of control cells (Figure S4).
We then used triple-quadruple mass spectrometry via selected reaction monitoring (SRM) to identify metabolite changes in Bcl-xL cells relative to GFP control cells as mass spectrometry is a more sensitive approach (Bajad et al., 2006; Lu et al., 2008). This is particularly relevant for intermediates of glucose metabolism as these metabolites are difficult to decipher by NMR due to their similar proton content. Thus, both NMR and mass spectrometry provide complementary approaches for a comprehensive understanding of the metabolite changes resulting from a specific perturbation. Indeed we found that acetyl-CoA levels were decreased by two-fold in Bcl-xL expressing cells relative to GFP expressing cells by mass spectrometry as well as an enzyme-based assay (Figure 5A–B). Conversely, acetyl-CoA levels were substantially increased in Bcl-xL−/− mouse embryonic fibroblasts (MEFs) compared to Bcl-xL+/+ MEFs (Figure 5C). These data provide strong evidence that Bcl-xL expression reduces the levels of acetyl-CoA, suggesting that reduced levels of acetyl-CoA in Bcl-xL overexpressing cells leads to hypoacetylation.
Since Bax/Bak DKO cells are not defective in protein N-alpha-acetylation, we reasoned that Bcl-xL might be able to negatively regulate the levels of acetyl-CoA independent of Bax/Bak binding. Cheng et al. reported that certain Bcl-xL mutants, such as F131V/D133A and G148E, are unable to bind to Bax or Bak but nevertheless retain 70–80% anti-apoptotic activity of WT Bcl-xL (Cheng et al., 1996). We measured acetyl-CoA levels in cells expressing WT Bcl-xL or these specific Bcl-xL mutants. A similar reduction in acetyl-coA levels was observed in cells expressing these Bcl-xL mutants and in cells expressing WT Bcl-xL (Figure 5D and 5E). Thus, Bcl-xL's metabolic function in regulating the levels of acetyl-CoA does not depend on its interaction with Bax/Bak.
As the majority of cellular acetyl-group in acetyl-CoA is produced from glucose (DeBerardinis et al., 2007), we asked whether glucose metabolism might be altered in Bcl-xL expressing cells. We fed Bcl-xL cells uniformly labelled 13C-glucose to differentiate glucose-derived metabolites from those derived from other carbon sources (Figure 6A; Table S1). We found that the levels of glucose-derived citrate were decreased by approximately 25% in Bcl-xL expressing cells relative to control (Student's t-test, p-value<0.05; Figure 6B; Table S1). As citrate is the direct precursor of cytoplasmic pools of acetyl-CoA, the lower levels of glucose-derived citrate might explain the decrease in acetyl-CoA levels observed in Bcl-xL expressing cells. Consistent with this hypothesis, levels of alpha-ketoglutarate, which is also derived from citrate, were lower in Bcl-xL expressing cells relative to control (Figure 6B; Table S1).
Since metabolite addition rescues the defect on protein N-alpha-acetylation by Bcl-xL (Figure 4A–B), we asked whether these metabolites could alter cell survival that is supported by Bcl-xL expression. Bcl-xL expression effectively protects against a wide range doses of doxorubicin (Figure 7A–D). Remarkably, increasing levels of citrate or acetate sensitized HeLa cells stably expressing Bcl-xL to doxorubicin-induced cell death compared to that of untreated cells (Figure 7B) with a two-fold increase in caspase activity (Figure 7D). Importantly, RNAi against acetyl-CoA synthetase or ATP citrate lyase completely suppressed the sensitization to doxorubicin elicited by addition of acetate or citrate, respectively (Figure 7E). This indicates that metabolite-induced apoptotic sensitization of cells expressing Bcl-xL specifically results from changes in acetyl-CoA production.
The above data suggest that Bcl-xL may mediate apoptosis resistance through two parallel pathways by inhibiting Bax/Bak oligomerization and by down-regulating protein N-alpha-acetylation. We therefore directly tested whether the effects of inhibiting Bax and ARD1 are additive in protecting against apoptosis. We found that double knockdown of both ARD1 and Bax indeed provided enhanced protection against apoptosis compared to that of knockdown individually, which was especially significant at higher concentrations of doxorubicin (Figure 7F). This finding supports the notion that Bcl-xL has dual functions in regulating protein N-alpha-acetylation levels and Bax/Bak oligomerization.
The ability to rapidly assess protein modifications immunologically has been essential for exploring the significance and regulation of multiple protein posttranslational modifications such as phosphorylation, histone methylation, and acetylation. Since an antibody for protein N-alpha-acetylation does not exist, the ability to assess this modification was severely limited. In this regard, the subtiligase assay as described in the present study provides a powerful tool to allow us to rapidly assess the endogenous levels of protein N-alpha-acetylation. Using this assay, we discovered that protein N-alpha-acetylation status is reduced in cells overexpressing Bcl-xL. Furthermore we show that protein N-alpha-acetylation is sensitive to acute changes in acetyl-CoA availability. Since incomplete N-alpha-acetylation status of proteins has been described by recent proteome studies (Arnesen et al., 2008; Goetze et al., 2009), we propose that protein N-alpha-acetylation might be a regulated process. In the course of our studies, we noted that protein N-alpha-acetylation levels as assessed by subtiligase were sensitive to nutrient levels in the culture medium (Yi CH and Yuan J, unpublished observations). Because ARD1-mediated N-alpha-acetylation requires acetyl-CoA as an essential cofactor, we hypothesized that protein N-alpha-acetylation is influenced by cellular metabolism.
Our study directly links a specific metabolite, acetyl-CoA, to apoptotic sensitivity and supports a growing number of studies that describe a role for cell metabolism in controlling apoptosis (Nutt et al., 2009; Nutt et al., 2005; Schafer et al., 2009; Vaughn and Deshmukh, 2008). Interestingly, the cellular levels of acetyl-CoA are sensitive to Bcl-xL status in a Bax/Bak-independent manner because the expression of Bcl-xL mutants that are unable to bind to Bax or Bak (Cheng et al., 1996) can also affect the acetyl-CoA levels to the same extent as that of wild type Bcl-xL. Hardwick and colleagues demonstrated that these Bcl-xL mutants preserve 70–80% anti-apoptotic activity of WT Bcl-xL despite their inability to bind to Bax or Bak (Cheng et al., 1996). Thus, inhibiting acetyl-CoA production might provide an additional mechanism for Bcl-xL to protect against apoptosis in a Bax/Bak-independent manner. Taken together, these data suggest that Bcl-xL might protect against apoptosis through two parallel mechanisms: by directly binding and inhibiting Bax/Bak oligomerization and by regulating mitochondrial metabolism, which leads to reduced levels of acetyl-coA and protein N-alpha-acetylation.
We propose that Bcl-xL integrates metabolism to apoptotic resistance by modulating acetyl-CoA levels. Previous studies show that Bcl-xL directly binds to the voltage-dependent anion channel (VDAC), a component of the mitochondrial permeability transition pore, which controls mitochondrial metabolite exchange (Shimizu et al., 1999; Vander Heiden et al., 2001). It is possible that Bcl-xL expression may alter levels of acetyl-coA by regulating mitochondrial membrane permeability. Citrate carrier (CiC), a nuclear-encoded protein located in the mitochondrial inner membrane and a member of the mitochondrial carrier family, is responsible for the efflux of acetyl-CoA from the mitochondria to the cytosol in the form of citrate (Gnoni et al., 2009; Kaplan et al., 1993). We found that the levels of glucose-derived citrate were decreased by approximately 25% in Bcl-xL expressing cells relative to control. This reduction in citrate levels could explain the observed decrease in acetyl-CoA levels in Bcl-xL expressing cells and contribute to anti-apoptotic function of Bcl-xL. Indeed, addition of citrate to Bcl-xL expressing cells leads to increased protein N-alpha-acetylation and sensitization of these cells to apoptosis. Perturbations in acetyl-CoA production may extend to other oncogenic contexts beyond that of Bcl-xL. For example, the levels of acetyl-CoA pools derived from glucose were found to be approximately 20% higher in myc +/+ cells relative to myc −/− cells (Morrish et al., 2009). The ability of myc to increase in acetyl-CoA levels might contribute to enhanced apoptotic sensitivity of cells overexpressing c-Myc (Evan et al., 1992). We propose that the basal levels of acetyl-CoA may influence the apoptotic threshold in multiple oncogenic contexts.
The ability of Bcl-xL to control the levels of acetyl-CoA and protein-N-acetylation provides a clear example by which metabolism is mechanistically linked with apoptotic sensitivity. Loss-of-function ard1 mutant yeast are specifically defective in alpha-factor response but not to a-factor (Whiteway and Szostak, 1985), indicating that protein N-alpha-acetylation status can dictate specific cellular behavior or process. Since protein N-alpha-acetylation affects a large number of cellular proteins, we propose that metabolic regulation of this process exerts its control on cellular processes through regulating a group(s) of proteins rather than individual proteins. Ard1 deficient mammalian cells are defective in the activation of caspase-2, caspase-3 and caspase-9 in response to DNA damage (Yi et al., 2007). Consistently, the N-alpha-acetylation of multiple caspases, including caspase-2, caspase-3 and caspase-9 was reduced in Bcl-xL overexpressing cells. It is possible that defects in N-alpha-acetylation of multiple caspases, which may negatively regulate their activation, contribute collectively to the apoptosis resistance in Ard1 deficient cells and Bcl-xL overexpressing cells. Thus, the levels of acetyl-CoA that control the N-alpha-acetylation of multiple proteins involved in a pathway may collectively determine the specific outcome of the cell fate. In this regard, the role of acetyl-CoA is clearly beyond what a cofactor normally serves: we propose that acetyl-CoA is a signalling molecule that provides an important liaison between metabolism and multiple cellular processes.
For RNAi studies, low passage HeLa cells were transiently transfected (5 × 105/well in 96-well plates) with a pool of four siRNAs (pre-designed ON-TARGETplus siRNAs available from Dharmacon, 50nM) using Oligofectamine transfection reagent (Invitrogen). Following a 48-hour incubation, cells were treated with doxorubicin (concentrations and incubation times noted in figure legends). siRNAs were tested in triplicate for each independent experiment. For detection of caspase cleavage by western blot, HeLa cells were transfected as described above (1.5 × 105/well in 6-well plates) followed by treatment with doxorubicin. Cells were lysed directly in SDS samples buffer and subjected to SDS-PAGE and western blot analysis using standard procedures.
For metabolite sensitization experiments, HeLa cells stably expressing GFP or Bcl-xL (3 × 105/well in 96-well plates) were pre-treated with acetate (50mM) or citrate (10m) for 24 hours followed by treatment with doxorubicin as indicated.
Cell viability was determined by measuring cellular ATP levels (CellTiter-Glo Luminescent Cell Viability Assay, Promega). Caspase-3/7 activity was quantified using a luciferin-labeled DEVD peptide substrate (Caspase-Glo 3/7 Assay, Promega). Luminescence was measured using a Wallac Victor2 plate reader.
The biotinylated peptide substrate for subtiligase with a TEV protease cleavage site, biotin-ahx-ahx-GGTENLYFQSY-glc-Y-NH2, was synthesized manually on a rink amide MBHA resin according to standard 9-Fluorenylmethoxycarbonyl (Fmoc) chemistry-based solid phase peptide synthesis protocols (Mahrus et al., 2008). The crude peptide was purified using a C18 semi-preparative reverse phase column on a Waters HPLC system. The identity of the purified product was confirmed by ESI-MS (isotopic MW calculated 1949.9 Da, m/z [M+H]+ found 1951.0).
The peptide substrate could be made more soluble by incorporating D-arginine residues into the sequence (Yoshihara et al., 2008). So a more soluble form of the substrate, biotin-ahx-ahx-dRdRdR-ahx-ahx-GGTENLYFQSY-glc-Y-NH2, was also synthesized, and its integrity was verified by HPLC and ESI-MS (MW calculated 2644.4 Da, m/z [M+H]+ found 2645.8).
The expression construct for subtiligase was prepared using the plasmid pBS42 (ATCC 37279) according to published procedures (Wells et al., 1983) except that a His6 tag was added to the C-terminus (Tan et al., 2007). The gene of the subtiligase variant contains point mutations S221C, P225A, M124L and S125A on wild-type subtilisin BPN′. The recombinant protein was expressed in B. subtilis RIK1285, which is deficient in the production of neutral and alkaline proteases. Purification of subtiligase was performed as previously described (Abrahmsen et al., 1991) except that a Co2+ affinity chromatography step was used instead of ion-exchange chromatography (Tan et al., 2007). The affinity-purified subtiligase was desalted using a PD-10 column (Amersham) with deionized H2O. Aliquots of the desalted enzyme solution were flash-frozen, lyophilized and stored at −80°C until utilized. The prepared subtiligase was analyzed by SDS-PAGE gel and MALDI-TOF MS, which confirmed its purity and identity. The ligase activity of the enzyme preparation was confirmed in model ligation reactions using known peptide substrates for subtiligase.
Cells used for subtiligase assay were plated at 50–60% confluency to support exponential growth followed by pre-treatment with acetate (50mM) or citrate (10mM) for 24 hours as indicated. Cells were lysed in 0.2% Tween 20 and 0.2% Triton X-100 buffer, and resulting lysates were used for subtiligase reaction that includes 1mM purified subtiligase, 1mM purified biotin-peptide, and 2mM DTT as previously described (Mahrus et al., 2008). Reactions were allowed to proceed for 1 hour at room temperature. Biotinylated proteins were affinity purified with Neutravidin agarose at 4° overnight (Thermo Scientific). The following day, agarose was pelleted and washed three times in lysis buffer. Purified proteins were eluted directly in 2X SDS sample buffer and eluants were analyzed by SDS PAGE.
3 replicates of Jurkat cells stably expressing GFP or Bcl-xL (5×106 for each replicate) were washed twice in PBS and resuspended in RPMI medium with glutamine, 10% dialyzed NCS, and 10mM uniformly labeled 13C-glucose (Cambridge Isotope Laboratories) followed by incubation for 24 hours. Cells were washed twice, and metabolites were extracted in 3mL 80% ice-cold methanol. Insoluble material in lysates was pelleted at 13,000g for 10 minutes and methanol from resulting supernatant was evaporated. Samples were resuspended using 20µL HPLC grade water for mass spectrometry.
7µL of 20µL were injected using a 5500 QTRAP mass spectrometer (Applied Biosystems/MDS Sciex) equipped with a Prominence UFLC HPLC system (Shimadzu) via single reaction monitoring (SRM) of a total of 249 endogenous metabolites for 12C analyses of GFP and BcL-xL samples. For analyses of 13C labeled GFP and BcL-xL samples, 153 endogenous metabolites were targeted via SRM. Reliable quantitative data are only acquired from approximately 60% of the targeted metabolites (~160 metabolites from the 12C method and ~55% from the 13C method (~80 metabolites). Some metabolites were targeted in both positive and negative ion mode including acetyl-CoA. ESI voltage was 5000V in positive ion mode and −4500V in negative ion mode. The dwell time was 5ms per SRM transition and the collision energy was optimized for each SRM transition. Total cycle time was 2.09 seconds for the 12C method and 1.24 seconds for the 13C method. Scheduled SRMs were not utilized. Samples were delivered to the MS via normal phase chromatography using a 2.0 mm i.d × 10 cm HILIC Luna NH2 column (Phenomenex) at 250µL/min at basic pH using positive and negative ion switching within the same 30 minute LC/MS/MS analytical run. Gradients were run starting from 85% buffer B (HPLC grade acetonitrile) to 42% B from 0–5 minutes; 42% B to 0% B from 5–16 minutes; 0% B was held from 16–24 minutes; 0% B to 85% B from 24–25 minutes; 85% B was held for 7 minutes to re-equilibrate the column. Buffer A was comprised of 20mM ammonium hydroxide/20mM ammonium acetate in 95/5 water/acetonitrile. All metabolomic measurements were performed in triplicate. Peak areas from the total ion current (TIC) for each metabolite SRM transition were integrated using MultiQuant v1.1 software (Applied Biosystems).
Statistical analysis was conducted using unpaired, two-tailed Student's t-test.
We are indebted to Alfred Goldberg, Tom Rapoport, Andrew Steele, Marta Lipinski, and Benedicte Py for critical reading of the manuscript, to Pere Puigserver, Harvey Lodish, Kristin White, and Danny Chou for helpful discussions and to Xuemei Yang for help in acquiring mass spectrometry metabolomics data. We thank Thomas Arnesen and Johan Lillehaug (University of Bergen) for anti-NATH as well as with help in designing caspase-2 N-alpha-acetylation mutants. We also thank Tullia Lindsten and Craig Thompson (U Penn) for providing anti-Bcl-xL as well as Bcl-xL +/+ and Bcl-xL −/− MEFs. VSV-RAIDD and Bcl-xL mutant constructs were generously provided by Jurg Tschopp (University of Lausanne) and Emily Cheng (Wash U), respectively. This work was supported, in part, by an Innovator's Award from the U.S. Army Breast Cancer Research Program (grant DAMD17-02-1-0403), a grant from the National Institute of Aging (R37-012859) and a NIH Director's Pioneer Award to J. Yuan, NIH grants (DK070299 and GM47467) to G. Wagner, NIH grant (5P01CA120964-03) to J.M. Asara, and a Kirchstein National Research Service Award from the National Institute of Neurological Disorders and Stroke (grant 1F31NS057872-01) to C.H. Yi.
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Week of May 24 – 31
No need to hit those freeways out of town this weekend. You can stay home and join in all the fun at the Bellaire Club with all your friends and the friends you have not met yet. You will be safe here.
Starting Sunday at 1:15, right after the monthly birthday meeting of the Sunday 12:15 Noon Meeting we will kick it off with the Grand Opening of the monthly Game and Cupcake Social. Guided along by board member, Jonathan L., the event will include games like Bingo, Chess, Dominoes, Checkers, Poker, Bridge or whatever game members attending can get going. We have plenty of room for plenty of games, so let's get this tradition started with a bang.
Come back Monday for either the Monday Men's Meeting or the Women's Health and Healing Meeting,both at noon. Following the meetings we will be treated to traditional fare like hot dogs, Frito pies andwatermelon plus anything else our creative cooks decide to bring. This gets us in the mood for a special Memorial Day speaker, veteran Jerry C. His is a colorful story you will not want to miss.
Following the speaker meeting will be more games of bingo or anything else you can get going.
Rick W. has for years now taken time off from his very busy career to lead a meeting at one of the nearby prisons on Friday afternoons. He always welcomes anyone willing to tag along to share their recovery with inmates eager to hear fresh voices from the outside world. To get involved, you must first be approved by the Texas Department of Corrections. Rick will handle this, but he will need your full name, date of birth, drivers license number and address as it appears on your license. To get the ball rolling, send this information to Rick at email@example.com and Rick will take care of the rest. All who have gone out to the meetings with Rick have reported it to be a most gratifying experience. Your voice will be appreciated.
- Beginning this week, various groups meeting at the Bellaire Club will celebrate the AA birthdays of those who sobered up this month. We congratulate all of them and hope them another successful year of sobriety.
- At the 10:00 AM Open Door Group. Greg W. will be celebrating five years sobriety.
- At the daily (except Saturday) 6:30 PM 12 & 12 Group one of the leaders, Ronald C. will celebrate 13 years
- Busiest group this month will be the Thursday Women's Emergency Room Group meeting at 6:30 PM where the celebrants will be Traci E. with seven years, Davasha S. with 12 years,Peggy S. with 20 years, Patty J. with 21 years, and B.J. McC. with 22 years sobriety.
- At Taco John's Saturday Noon Meeting, Mark W. will be celebrating his first year of sobriety
- At the Sunday Noon Group meeting at 12:15, Davasha S. will celebrate 12 years, Jeff T., 17 years, Peggy S., 20 years and Andrew E., 24 years.
- At the Wednesday 6:30 AM Red-Eye Special Meeting, Baroque F. will celebrate one year of sobriety
- Our congratulations to all of these celebrants of major milestones in their sobriety.
If you are looking for a nice gift to give any of the celebrants, may we suggest making a donation in their honor of any amount you choose to the Bellaire Club so instrumental in their sobriety? That gift will be most appreciated by the celebrants as well as the club. Ask anyone behind the counter for a card.
- The 12 & 12 Group that meets daily except Saturday has this roster of leaders. Thursday will be Ryan; Friday, Julio; Sunday, Ronald; Monday, Johnny M.; Tuesday, Lawauna S. and Wednesday, Ralph.
- Jeff T. will be chairing and leading the Thursday Noon Meeting.
- Chairing The Thursday Women's Emergency Room Group this month is Ann N. who has invited Amy D. to be the leader. The meeting starts at 6:30 PM.
- The Friday Two Dollar Meeting that meets at 12:15 is being chaired this month by Jane E.and Davasha S. will lead the meeting this Friday.
- Don't forget Taco John's regular Saturday Noon Meeting.
- The group formerly known as the Saturday Speakers Meeting at 6:30 PM now is an Open Discussion Meeting with a speaker the last Saturday of the month and is in search of a new name. Several have been suggested. Drop by and offer yours.
- The 11:00 AM. Sunday Speaker's Meeting being chaired by Doug DeB. will have Jim H. as the speaker this Sunday. With 36 years sobriety, Jim can be found at the West Chase Nooners Group and the Bunker Hill Tuesday Night Group as well as the Sunday Speaker's Meeting.
- Arch A. will lead the special monthly birthday celebration of the Sunday Noon Meeting this Sunday at 12:15.
- The Monday Noon Hope and Healing Women's Group is being led this month by Alice G. Next month Koi D. will take the lead.
- And over on the men's side, at The Monday Men's Meeting, Larry A. or Barry K. are manning up and leading the group.
- Rick W. and Robert P. will continue to chair the lively Blankety-Blank It's Tuesday Noon Meeting
- The new and well receivedThe Red-Eye Special Group thatmeets every Wednesday at6:30 AM, is celebrating its fifth month with Larry A. and Robert P. there to guide it along.
- Robert P. will chair the Wednesday Noon Meeting, the Where-U-At Group and has announced that Will G. will be the leader.
For a complete list of all meetings held at the Bellaire Club, please go to our website bellaireclub.org and click on Meeting Schedule.
- Be content with what you are and wish not change; nor dread your last day nor long for it.Marcus Aurelius
- Better to fight for something than live for nothing. George S. Patton
- Age wrinkles the body. Quitting wrinkles the soul. Douglas MacArthur


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|December 5, 2012, 10:33 PM
Junior Member
Join Date: July 5, 2012
Posts: 7
Barrel Nut Question
How hot does the barrel nut on an ar15 get after say a mag dump? I ask because I have the Magpul MOE handguards and they have some play in them, but adding electrical tape to the side that contacts the teeth of the barrel nut removes the play entirely. I dont want the tape to melt or catch fire and for there to be a sticky mess in my handguards.
|December 5, 2012, 11:24 PM
Senior Member
Join Date: April 10, 2012
Location: San Diego CA
Posts: 3,853
I use electrical tape all the time at work ( construction ) . I've noticed just moderate heat can make electrical tape get gummy and want to slide on it's self . Not sure that's your best option . I do not have a recommendation but I would not recommend elec tape . I'm sure somebody here has had this problem and has a great fix .
Tolerate- allow the existence, occurrence, or practice of something that one does not necessarily like or agree with , without interference.
If you have some time IMO this is worth a listen/watch but it takes a few minutes to really get going .
https://www.youtube.com/watch?v=USg3NR76XpQ&t=3265s or a picture of Mohamed https://www.youtube.com/watch?v=1VwpwP_fIqY
|December 6, 2012, 03:54 AM
Senior Member
Join Date: November 22, 2006
Posts: 789
I've tightened up wobbly handguards before by wrapping Teflon tape around the barrel nut. It's got a high heat resistance, is not adhesive, and you can wrap it slowly until you get your desired level of tightness on the handguard.
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Shutter Island by Leonardo Di Caprio seems to be a really great movie. I watched the trailer in Youtube and it was awesome. The movie is a potpourri of horror and suspense. I look forward to watching the movie as soon as possible... as soon as its available in my place.
by Dale Mazurek7 years ago
Well yesterday my wife and I went to see the new release The Crazies.Anyways I am not a movie reviewer but thought I would try a little.On a scale of one to ten I would give the movie a fourYou can see all the scary...
by Nick17Pierce197 years ago
I've heard alot of people saying this was a great film, just not what they expected. Who all out there has seen the movie? What did you think?
by Cuselax7 years ago
Just curious, did anyone see the movie inception?
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text: Hi friends.. Today i am going to tell you about how to insert data available in HTML from using PHP in to MySQL database. Here i will use post method for transfer data using HTML form to my PHP script. Lets see here how to create form without having table. I mean you can create… Continue reading How to insert data to MySQL using PHP


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MOVIE SYNOPSIS [ General Audiences ] Under the unique direction of Michael Schwarz and Edward Gray, this film with audio in English (original language) was premiered in 2009. The movie was produced in USA. You can watch The Botany of Desire online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2009.
Rating: 5 by 358 users./
Runtime: 120 minutes
Frances McDormand and Michael Pollan.
120-minute film (full movie) directed by Michael Schwarz and Edward Gray.
The original audio of The Botany of Desire has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2009.
Kate McLean, Cianna Pamintuan Stewart and Edward Gray.
This film was produced and / or financed by Kikim Media.
Composed by Martin Bresnick.
Public Broadcasting Service (PBS), PBS International and PBS Distribution.
Without commercial breaks, the full movie The Botany of Desire has a duration of 120 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, The Botany of Desire in FULLTV Movies.


text: You can order directly from the JM Spreadshirt Shop and choose from many styles, colors and sizes of JM merchandise, including dog bandanas! It doesn't cost us anything and a portion of every sale comes back to the PTA. We'll be selling physical merchandise at our Joaq-a-thon 2017, so you can also buy from us in person.As always, thanks for the support and showing your JM pride!