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Selection of nokia
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subset: commoncrawl


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WW 576 arrives at Dividing Creek and will soon do some switching at a nearby siding. The train is passing a private collection of ALCOs which includes fomer LIRR "power pack" FA2s and a... (more)
After crossing Lummis Mill Road, the engineer running WW 576 will notch out the throttle. The solo GP10 sounded great digging into the slight grade with 33 empty sand hoppers in tow, and the adde... (more)
A WW crew builds a consist of empty sand cars at Bridgeton before heading south for Dividing Creek. This train has a GP10 at each end of the train to facilitate switching moves. WW 732 is a rece... (more)
WW 576 switches out sand cars at a siding along the aptly-named Railroad Avenue. This was formerly the CNJ Cumberland & Maurice River Branch. The line was purchased from Conrail by the WW in 198... (more)
Winchester and Western 445 leads a small Bridgeton bound local south down the Ex CNJ Southern Division on a beautiful summer day.
Three GP9s and a GP10 power 86 south through Martinsburg after working the CSX interchange.
Winchester and Western Hagerstown local heads back to Gore VA after interchanging with the Norfolk Southern in Hagerstown. Seen passing Gowers feed mill in Williamsport , MD
Winchester & Western Local comes to Hagerstown Maryland to interchange with the Norfolk Southern
After dropping off 11 cars at the CSX interchange, 86 continues on passing over the rebuilt right of way over CSX and Tuscarora Creek before heading into downtown M... (more)
For this and other photos please visit my site at http://... (more)
Fund on a Hyatt built EMD 6 1/2 by 12 roller bearing.
For this and other photos please visit my site at more)


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Sharp Analog Wall Clock
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text:
Jack Cunningham wants to
Remove Obama From Office If He Bypasses Congress On Anmesty
Obama Violates Our Constitution
Obama Violates Our Constitution
Too many child abusers, murderers are getting off too easy.
Because this is horrendous and hideous. Can you imagine the fear these animals must be in the last minutes of their
Belivers should sign to let the lawmakers and the so called powers that be know we should be allowed to openly pray, and
Build a advanced Culture of Compassion and Empathy for Humans,Nonhumans,Earth and


text:
Title: War Stories
Author: babies stole my dingo (agilebrit)
Fandom: Buffy the Vampire Slayer
Rating: PG-13 for mention of sex.
Length: Short short (under 1000 words)
Disclaimer: Joss is the genius behind these characters; I am but a lowly follower. I make no money from any of this, so please don't sue me.
Written for: AppomattoxCO requested a Giles&Anya fic, set during a slow day at the Magic Box, during which they trade war stories.
"Bugger all, Anya, would you stop prowling about, please? You're driving me quite mad," Giles said, looking up from a big leather-bound book.
The Magic Box was absolutely dead. They hadn't had a customer all day, and Anya wandered around the shop, dusting, picking things up and putting them down again, and sighing loudly at regular intervals.
"Well, I'm sorry, Giles. It's not my fault the customers have gone poof and I don't have anything to do."
He took his glasses off and rubbed the bridge of his nose. "Well, it's nearly lunchtime. Shall we order out for something? Chinese? Pizza?"
"I haven't had pizza in awhile. Let's call Pete's."
After it was delivered, they sat at the research table. "Mmm, this is good," Anya said through a mouthful of cheese and sausage. "You know--" She swallowed. "--the pizza guy reminds me of one of the unfaithful men I was summoned to do vengeance on, back in the day. What was it he did?" She took another bite, musing. "Something dumb, like sleeping with the Maid of Honor the night before the wedding. Oh, and she got pregnant. His wife was quite unhappy about that."
"I'm almost afraid to ask what you did to him."
"She was really creative. We tied him up and fed his left testicle to a piranha, and his right one to a Chihuahua, while he watched. He thought fish were boring, and he hated little yappy dogs. We gave him a reason to hate the dogs and taught him that he was wrong about fish. And then she wished for him to have a disease that made his penis rot off." Bite, chew. "Good times."
Giles carefully put his pizza slice down. "Remind me to never upset you."
"Don't be silly. I couldn't do anything like that to you, Giles. Well, not unless you made me really angry. It's not like I have any powers anymore anyway." She waved her pizza in the air. "What about you? Ever do anything wild and crazy in your errant youth? You must have."
He smiled a little. "There was this time--" He stopped abruptly. "That is neither here nor there and completely irrelevant to my current station in life."
"Your current station in life," she said, munching, "is that of a lonely, stuffy, expatriate Englishman, who hasn't had an orgasm in years, running a magic shop with no customers."
"Yes, thank you so much for the reminder."
"Come on, Giles, tell." She grinned mischievously. "If you don't, I'll start prowling around again."
"Well, we can't have that, can we? All right. One of my classmates at Oxford was a bit of a bully. He was on the rowing team and got away with bloody everything. His girlfriend was an absolute little cow who came from money. One night, Ethan Rayne and I turned them into trolls in the middle of an--" He coughed. "--intimate moment." At the same time, Giles and Anya chorused, "Eelsbane." Giles choked back a laugh. "We changed them back the next day, and let them know that messing about with us anymore was likely to result in effects that were more...lasting."
"Not bad vengeance for a couple of amateurs," Anya judged. She eyed him. "You wouldn't do anything like that to Xander and me, would you?"
"Certainly not." He lifted his brow. "Unless I somehow needed to prove that I'm not as stuffy as you lot seem to think I am."
Now it was her turn to put her pizza down with a gulp, just as the bell above the door rang. She gave Giles a bright smile and left to help their first customer of the day, while he cleaned up the remains of their lunch and reflected that maybe they weren't so different, after all.


text:
Eating Stone
Eating Stone
Imagination and the Loss of the Wild
Vintage Books USA, Paperback, 9781400031771, 330pp.
Publication Date: October 17, 2006
"Piercingly beautiful. . . . Its chapters map a vibrant, curious mind in love with the particulars of the Southwest landscape." –The New York Times Book Review"One of our finest natural-history writers. . . . Her own knowledge of the natural world is deep, her prose breathtakingly beautiful and often startling." –Annie Proulx, Globe & Mail"A major contribution to an understanding of the land. . . . Meloy's genius seems evident on every page of this thoughtful, impressionistic book."–Deseret News "One of the American West's greatest contemporary naturalists. . . . More than a mere adventure, Eating Stone concludes Meloy's love affair with the western desert and the wildlife it nourishes."–Outside MagazineBeautiful. . . . Not since Peter Matthiessen's The Snow Leopard has an author transported us so completely into the wilderness."–The Plain Dealer


text:
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Luke Cover #119
Leia Cover #119
Han Cover #119
Star Wars Insider #120 Subscriber's cover
Star Wars Insider #120 "War Stories" Cover
©2016 Twitpic Inc, All Rights Reserved


text:
See My Wife - March 3, 2008
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How could we call ourselves the Kings of porn if we didn't have a nice blonde MILF like Emilianna in our arsenal of sex stars? We pride ourselves on bringing the best porn on the Internet right to you and Emilianna is one of our stars that is sure to make that happen. Emilianna has done it all. Group sex, girl on girl, guy on girl, interracial, anal, oral, if you think it she will fuck it. In particular, Emilianna likes to have group sex so she can play with as many girls and guys as she wants and the sex can last for hours. See her best performance at the pool party gangbang orgy in the Coochie Crew episode of the VIP Crew website. You'll love this MILF!
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text:
Dr. Alvin SayersOctober 23,2013Dr. Alvin Sayers
FAIR HAVEN Dr. Alvin Sayers, 83, died Tuesday, Oct. 22, 2013, at Rutland Regional Medical Center following a long illness.
A complete obituary will be published in a future edition of the Rutland Herald.
Arrangements are with the Durfee Funeral Home in Fair Haven.MORE IN ObituariesAnthony J. Full StoryHarris Peel
DANBY Harris Peel, 92, of Danby, died Thursday, Sept. Full StoryAlberta Lamb-Rogers
ELMIRA, NY Alberta Lamb-Rogers (nee McAuley), Age 99, of Elmira,... Full Story
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AP Photo/Brian Kersey
Jared Sullinger commits a foul vs. the Bulls earlier this season.Even though he's played well beyond his years at times this season, NBA officials have constantly reminded Jared Sullinger that he's still only a rookie.
Sullinger has been whistled for 74 personal fouls this season, more than any other member of the Celtics (Brandon Bass checks in at No. 2 with 67), and is currently averaging six fouls per 36 minutes. He fouled out for the first time in his career during last Friday's loss to the Bucks, and has finished with three or more fouls in 16 of 26 games this season.
Among NBA rookies, Sullinger ranks fourth in total fouls, behind Kyle Singler of the Pistons, Jonas Valanciunas of the Raptors, and Tyler Zeller of the Cavaliers -- all of whom average more minutes than Sullinger.
While Sullinger has certainly been deserving of his fair share of whistles, it doesn't seem like a stretch to point out that he's also been on the receiving end of a number of questionable calls on straightforward defensive plays, charge/block scenarios, and even as he's battled for rebounds. Consider Friday's loss in which, during the second quarter, Sullinger secured a rebound, brought the ball just below with his chin with his elbows out, and was immediately whistled for a foul for trying to clear space to make an outlet pass.
Plays like those are fairly routine in the NBA, which is why Sullinger, head coach Doc Rivers, and even Kevin Garnett, who was mere feet away from Sullinger at the time, couldn't help but express their disbelief over the call.
The unfortunate reality is that Sullinger is simply enduring rookie calls. Refs aren't overly familiar with Sullinger's game, and because he's still unproven, if he finds himself in a situation where there is questionable contact with another player, officials are more likely to crack down on him because of his first-year status.
To his credit, Sullinger hasn't lashed out at officials after questionable calls this season, nor has he willingly voiced his displeasure to the media. He's realistic about the calls he's been getting, and he understands that, sometimes, it's just a part of the rookie package.
"It is what it is. It's fouls. I can't go over to the ref and yell at him and make him change the call," Sullinger said. "Once he makes a call, he makes a call, so it goes in one ear and out the other.
"I'm a rookie. There's no way I can handle a ref. They're not going to listen to me. So, being a rookie, sometimes you always get the short end of the stick. But you've got to pay your dues around here in this league."
One thing Sullinger has had a habit of doing, though, is extending his arms out in an incredulous fashion after a suspect whistle. As a look of disbelief crosses his face, the arms shoot out, the palms go up, and Sullinger can only be left wondering what he did (or didn't do) to earn the call. Consider it the mild-mannered cousin of Kendrick Perkins' patented scowl after a whistle.
Sullinger did concede after Sunday's practice that he does resort to the arms-out reaction pretty frequently.
"I probably do lead the league in arms out after fouls, but it is what it is," Sullinger said.
Things are unlikely to change for Sullinger on this front moving forward. It's a part of his current NBA reality that he needs to begrudgingly accept. Assuming Sullinger can continue to make strides and contribute this season and in future seasons, officials should eventually learn his game and be a little more lenient with their whistles. But for now, all Sullinger can do is continue to play through the calls and hope that he'll eventually earn the benefit of the doubt from the referees further down the road.


text:
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Conceived and designed the experiments: XW. Performed the experiments: XW YH ZFL. Analyzed the data: XW YH. Contributed reagents/materials/analysis tools: XW. Wrote the paper: XW. Helped write the paper: QL.
Alpha-dystroglycan (α-DG) is a ubiquitously expressed receptor for extracellular matrix proteins and some viruses, and plays a pivotal role in a number of pathological events, including cancer progression, muscular dystrophies, and viral infection. The O-glycans on α-DG are essential for its ligand binding, but the biosynthesis of the functional O-glycans remains obscure. The fact that transient overexpression of LARGE, a putative glycosyltransferase, up-regulates the functional glycans on α-DG to mediate its ligand binding implied that overexpression of LARGE may be a novel strategy to treat disorders with hypoglycosylation of α-DG. In this study, we focus on the effects of stable overexpression of Large on α-DG glycosylation in Chinese hamster ovary (CHO) cell and its glycosylation deficient mutants. Surprisingly, stable overexpression of Large in an O-mannosylation null deficient Lec15.2 CHO cells failed to induce the functional glycans on α-DG. Introducing the wild-type DPM2 cDNA, the deficient gene in the Lec15.2 cells, fully restored the Large-induced functional glycosylation, suggesting that Large induces the functional glycans in a DPM2/O-mannosylation dependent manner. Furthermore, stable overexpression of Large can effectively induce functional glycans on N-linked glycans in the Lec8 cells and ldlD cells growing in Gal deficient media, in both of which circumstances galactosylation are deficient. In addition, supplement of Gal to the ldlD cell culture media significantly reduces the amount of functional glycans induced by Large, suggested that galactosylation suppresses Large to induce the functional glycans. Thus our results revealed a mechanism by which Large competes with galactosyltransferase to target GlcNAc terminals to induce the functional glycans on α-DG.
Alpha-dystroglycan (α-DG), a highly glycosylated plasma membrane-associated protein, was originally isolated from brain and skeletal muscle [1], [2]. It is encoded by DAG1 gene and expressed ubiquitously [3]. The DAG1 gene is translated as a single polypeptide which is post-translationally cleaved into two subunits: α-DG and β-DG. The two subunits associate non-covalently as the key components of the dystrophin glycoprotein complex (DGC) [4]. Alpha-DG associates with extracellular matrix (ECM) proteins, while the transmembrane β-DG interacts with the sub-membrane dystrophin or utrophin, which is, in turn, linked to actin-based cytoskeleton. Proper glycosylation of α-DG is essential for its binding to the ECM proteins such as agrin, laminins, neurexin, and perlecan. The linkage between ECM and the cytoskeleton through DGC is critical for the membrane integrity and functions of skeletal muscles. Alpha-DG consists of three distinctive domains, an N-terminal globular domain, a central mucin domain, and a C-terminal globular domain. The mucin domain (317–488aa) has a cluster of 50 Ser/Thr residues, which are potential sites for O-glycosylation. Removing the O-glycans on α-DG abolishes its ligand binding, indicating that the O-glycans are essential for the activity [4], although the exact structure of the O-glycans mediating its ligand binding remains largely unknown. The importance of the O-glycans on α-DG has been illustrated by the discoveries that the mutations in known and putative glycosyl-transferase genes such as POMT1/2, POMGnT1, LARGE, Fukutin, and Fukutin-related protein (FKRP) cause aberrant O-glycosylation of α-DG and result in various muscular dystrophies with a wide spectrum of clinical manifestations (termed as dystroglycanopathies) [5], [6], [7], [8], [9], [10]. The hallmark of these diseases is the hypoglycosylation of α-DG and reduced binding of the α-DG to laminin. However, among these genes, only POMT1/2 and POMGnT1 have been demonstrated to have the glycosyltransferase activities in the protein O-mannosylation pathway [6], [11]. The roles of LARGE, Fukutin, and FKRP in glycosylation of α-DG remain to be defined.
Human LARGE was originally identified as a tumor related gene with deletion in meningioma [12]. It is a type II transmembrane glycoprotein with 756 amino acids, residing predominantly in the Golgi apparatus [13]. Its N-terminal and C-terminal domains have sequence similarities to bacterial α-glycosyltransferase and mammalian β-1,3-N-acetylglucosaminyl-transferase, respectively. Despite the fact that glycosyltransferase activity of the LARGE gene has not been demonstrated, accumulating evidence suggested that LARGE plays a critical role in biosynthesis of the functional glycans of α-DG, which can be detected by immuno-staining with the IIH6 and VIA4 monoclonal antibodies and laminin binding assays. Significantly, aberrant glycosylation of α-DG is frequently associated with a variety of tumors, which have LARGE expression silenced [14], [15], [16], [17]. Interestingly, Campbell and colleagues reported that transient overexpression of LARGE can functionally bypass distinct genetic defects in myoblasts derived from patients with mutations in FCMD, POMT1, and POMTGnT1 [18]. Furthermore, previous studies also reported that transient expression of LARGE induced abundant functional glycans in the B421 and Lec15.2 cells with O-mannosylation defects, suggesting that transient overexpressing LARGE may process non O-mannosyl glycans to induce the functional glycans on α-DG [19], [20], [21], [22]. However, whether Large-induced functional glycosylation of α-DG is O-mannosylation dependent in physiological conditions remains unknown. Overexpressing LARGE can bypass hypoglycosylation of α-DG caused by non LARGE defects, suggested that overexpression of LARGE could be a potential therapeutic strategy for patients with hypoglycosylation of α-DG. However the detailed mechanism by which LARGE induces the functional glycans remains to be elucidated.
In the present study, we have utilized CHO cells and its glycosylation deficient mutants: Lec1, Lec2, Lec8, Lec15, and ldlD cells [23] and established an array of the cell lines with stable overexpression of mouse Large. With the O-mannosylation null deficient Lec15.2 cells, we demonstrated that stable overexpression of Large induces the functional glycans in a dolichol phosphate-mannose biosynthesis regulatory protein 2 (DPM2)/O-mannosylation dependent manner, thus confirming for the first time that Large is involved in modification of O-mannosyl glycans on α-DG. Quantitative analysis of the glycans on α-DG in the Lec8 and ldlD cells expressing Large revealed that Large can effectively induce the functional glycans on N-linked glycans. In addition, supplement of Gal to the conditioned media of ldlD-Large cell culture significantly reduced the amount of functional glycans, suggesting that galactosylation suppresses Large to generate the functional glycans. Our results revealed a mechanism by which Large competes with galactosytransferases to target GlcNAc terminals to generate the functional glycans on α-DG.
Previous reports on Large function were all carried out with transient overexpression, which often have extremely high but unsustainable levels of Large expression [19], [20], [21], [22]. To investigate the effect of stable overexpression of Large on specific glycans, we have established an array of glycosylation deficient CHO cell lines (Pro5, Lec1, Lec2, and Lec8), in which mouse Large tagged with a MYC epitope was stably expressed and termed as Pro5-LG, Lec1-LG, Lec2-LG, and Lec8-LG, respectively. The Lec2 and Lec8 cells are deficient in the Golgi CMP-sialic acid transporter and UDP-Gal transporter, respectively [23]. Consistent with previous reports with transient Large overexpression, stable overexpression of Large in both cells induced abundant functional glycans on α-DG as determined by the IIH6 monoclonal antibody, which recognizes the epitope(s) overlapping with laminin binding glycans on α-DG. Glycans on α-DG recognized by both the antibodies and laminin binding assay have been widely referred to as the functional glycans [24]. Similarly, the functional glycans were produced abundantly in the Lec1-LG cells deficient in enzyme MgalT1, which is essential for complex N-glycan formation on proteins [23]. The biological functions of the Large-induced functional glycans on α-DG in these cells were further supported by their cell surface localization examined by immuno-fluorescent microscopy with the IIH6 antibody (Fig. 1B) and the laminin cluster assay (Fig. 1C). These results are consistent with previous observation using transient expression methodology (Fig. 1, ,2),2), suggested that stable overexpression of Large induced the functional glycans independent to Gal, sialic acids, and complex N-linked glycans.
Large has been thought to modify O-mannosyl glycans to produce the functional glycans on α-DG to mediate its ligand binding. However, so far there has been no direct experimental evidence to support the hypothesis. To investigate the role of Large in o-mannosylation of α-DG, we established a stable cell line with Lec15.2 cells, in which Large was stably expressed. The Lec15.2 cell is deficient in DPM2 that plays an essential role in the complex of DPMs to produce dolichol-phosphate-mannose (Dol-P-Man). The Dol-P-Man is the substrate for enzymatic complex of POMT1/2 to initiate O-mannosylation of α-DG by adding the first mannose to Ser/Thr residues of α-DG in the lumen of ER [11]. Surprisingly, stable overexpression of Large failed to induce significant amount of the functional glycans in the Lec15.2 cells. The amount of the functional glycans was undetectable in the lysates from the Lec15.2-LG cells with the IIH6 antibodies, while its Large expression level is compatible to that in the other CHO-LG cell lines (Fig. 1A). The lack of the functional glycans on the Lec15.2-LG cells was further confirmed by the immuno-staining and laminin clustering assay (Fig. 1B, C). The results are apparently in contradiction to the previous reports in which several groups showed that transient overexpression of Large induced abundant functional glycans in either the B421 or LG 15.2 cells which are both deficient in the DPM2 gene [19], [20], [21], [22]. The Lec15.2-LG cells used in this study were selected from a large population of Lec15.2 cells. One possibility is that the selected Lec15.2-LG clones might have acquired additional genetic defects which may compromise the activity of Large. To address this issue, we independently repeated Large transfection of the Lec15.2 cells (3 times) and screened 1,000 clones overexpressing Large by indirect immuno-fluorescent microscopy. About 10% of transferant cells were capable of producing detectable IIH6 reactive glycans on the cell surface within 4-6 weeks after transfection. However, majority of the Lec15.2-LG clones gradually lost the capacity to induce the function glycans after 20 passages in continuing cell culture for 4 months (Fig. 2) despite the fact that the levels of Large expression are compatible to that in other tested CHO-LG cell lines (Fig.1 and and2).2). Four Lec15.2-LG clones retained the capacity of inducing functional glycans, but at much lower levels when compared to the other CHO-LG cells including Lec2-LG, Lec8-LG, and B421-LG cells. Three of the 4 IIH6 positive clones (Lec15.2-LG) were further examined by Western blotting for the amount of functional glycans and levels of Large expression together with Pro5-LG, B421-LG, and negative controls (Fig. 2). For comparison, the B421 cells were also transfected with the same Large expression construct and a number of clones were selected. In contrast to the Lec15.2-LG clones, all the B421-LG clones stably overexpressing Large maintained production of abundant functional glycans after 4 months culture (the same period for Lec15.2 clones), although the amount of functional glycans were about 50% of that in Pro5-LG cells (Fig. 2). The expression levels of Large in these B421-LG cells were similar to other CHO-LG stable cell lines including the Lec15.2-LG clones by the Western blotting assay. The consistent results between independent Large transfection experiments suggested that additional genetic defects are unlikely the reason responsible for loss of Large activity in the Lec15.2 cells.
To further address whether the deficiency of DPM2 is responsible for the loss of the Large function in the Lec15.2 cells, we conducted complementation experiments by introducing a wild-type mouse DPM2 cDNA into the Lec15.2-LG cells. In the experiments, the Lec15.2-LG cells were transfected with the DPM2 mammalian expression construct, and then examined by Western blot and laminin overlay assays (Fig. 3A). The DPM2 transfection restored Large function in the Lec15.2-LG cells, while DPM2 expression alone did not produce detectable the functional glycans. Furthermore, we also overexpressed DPM2-GFP in the Lec15.2-LG cells and examined functional glycans on the cell surface. Indeed, the expression of DPM2-GFP restored the Large function to produce the functional glycans in the Lec15.2-LG cells, while expression of DPM2-GFP alone failed to do so in either the Lec15.2 or the Pro5 cells (Fig. 3B). The results suggested that stable overexpression of Large induces the functional O-glycans on α-DG to mediate laminin binding in a DPM2/O-mannosylation dependent manner.
It has been reported that transient overexpression of Large modifies complex N-glycans on α-DG, particularly, in the Lec8 cells [19]. To investigate whether stable overexpression of Large has effect on N-linked glycans of α-DG as well, we used PNGase F to remove the N-linked glycans on α-DG from the Pro5-LG, B421-LG, Lec15.2-LG, Lec8-LG and ldlD-LG cells growing in the complete media (Fig. 4). The reduction of IIH6 staining signal in Pro5-LG, B421-LG, and Lec8-LG represents the amount of functional glycans on N-linked glycans and the remaining functional glycans would be on O-glycans. In the Lec15.2-LG cells, the remaining functional O-glycans would be on mucin type of O-glycans only since the cells are lack of O-mannosyl glycans due to missing DPM2 activity. Interestingly, removing N-linked glycans did not reduce significant amount of functional glycans on α-DG in all tested stable cell lines, except the Lec8-LG cells. The remaining functional glycans after PNGase F digestion was reduced to about 50% of that of untreated samples from Lec8-LG cells, suggesting that stable overexpression of Large only modifies N-linked glycans on α-DG to induce the functional glycans effectively in the galactosylation deficient Lec8 cells.
The structure of Large-induced functional glycan is unknown; and there is no method available to selectively remove O-mannosyl or mucin-type of O-glycans. Thus it is difficult to study the effects of Large on O-mannosyl and mucin-type O-glycans, separately. To circumvent this hurdle, we took advantage of the reversible glycosylation deficient CHO cells, ldlD cells, which are deficient in UDP-galactose 4′-epimersase (GALE) and produce neither UDP-GalNAc nor UDP-Gal resulting in pleiotropic glycosylation pathway defects [25]. Supplement of GalNAc and Gal to the cell culture media can restore the glycosylation pathways due to a salvage pathway in the CHO cells (Fig. 5A) [25]. Thus the mucin-type O-glycan biosynthesis could be manipulated by supplementing the sugar(s) to the cell culture media. In the experiments, the stable ldlD-LG cells were grown in the media with 3% lipoprotein deficient serum for 48 hours to deplete the intracellular UDP-GalNAc and UDP-Gal pools [25]. Under this culture condition the ldlD cells do not produce detectable mucin-type O-glycans by radioisotope labeling methodology [25]. We examined the amount of the functional glycans produced in the ldlD-LG cells growing in the cell culture media with different sugar supplements. Strikingly, the ldlD-LG cells produced the least amount functional glycans when grown in the medium supplement with both Gal and GalNAc, despite the glycosylation pathways were restored and evidenced by the β-DG immuno-blotting (Fig. 5). In contrast, the cells produced the most abundant functional glycans when supplemented with GalNAc alone. The comparison of the amount of the functional glycans produced in the ldlD-LG cells growing in different culture media [GalNAc (2.4) > non sugars (1.8)> Gal (1.6)>GalNAc and Gal (1)] is shown in Fig. 5B,C. To further analyze the effects of Large on N-linked and O-linked glycans quantitatively in the ldlD-LG cells, we removed the N-glycans from the α-DG proteins with PNGase F, which were harvested from the conditioned culture media with or without GalNAc (Fig. 5D, E). The results showed that without supplement of Gal and GalNAc, ~35% of the Large-induced functional glycans were on N-linked glycans (digested portion) and ~65% were on O-mannosyl glycans (remaining portion), since the mucin-type of the O-linked glycans are expected to be minimal. Supplement of GalNAc alone increases the total functional glycans by about ~28% comparing with that in the no sugar addition and about 25% of the functional glycans are on N-linked glycans (Fig. 5D,E).
In this study, we demonstrated that Large, a putative glycosyltransferase, induces the functional glycans on α-DG to mediate laminin binding in a DPM2/O-mannosylation dependent manner. Stable expression of Large failed to induce the functional glycosylation on α-DG, but it can be restored by expressing DPM2 in the Lec15.2-LG cells. Thus our results confirmed for the first time that Large is involved in the biosynthesis of the O-mannosyl glycans suggested by the results from clinical and mouse genetic studies. This result is also in-line with the recent report that the functional glycans may be linked to the first mannosyl residue of the O-mannosyl glycans on mucin domain [26]. It was curious to learn that the discrepancy between the results of the effects of stable expressing Large on α-DG glycosylation in the two Lec15 clones, B421 and LG15.2 cells. It can be explained that the B421 cells harbor the point mutation form of DPM2 which may allow the cells to produce sufficient amount of Dol-P-Man to support the function of stable expressing Large to produce abundant functional glycans. But the Lec15.2 cells produce neither DPM2 message RNA nor Dol-P-Man that is essential substrate for POMT1/2 complex to initiate the biosynthesis of O-mannosyl glycans in the ER lumen, thus resulting in loss of the Large activity in the Lec15.2 cells. On the other hand, when transient overexpression at extremely high level Large can produce the functional glycosylation of α-DG in the Lec15.2 cells (data not shown), suggested that Large can induce the functional glycans on N-linked and mucin O-glycans to bypass defects in the early steps of O-mannosylation pathway. But it can not be sustained.
Our results obtained from the sugar addition experiments with ldlD-LG cells clearly suggested that Gal inhibits the ability of Large to induce the functional glycans on the N-linked and mucin-type O-glycans. The mucin-type O-glycans consist of four components: Sia, Gal, GlcNAc, and GalNAc. Sia, Gal, and GalNAc are not the essential components of the functional glycans and addition of Gal or both Gal and GalNAc together even inhibited the functional glycosylation induced by Large. Thus only the GlcNAc could be the Large's target on mucin-type of O-glycans. This also explains that Large induces the functional glycosylation on N-glycans effectively in galactosylation deficient cells (Lec8) since the GlcNAc terminals are enriched on the N-glycans in galactosylation defective condition. Our results suggested that Large-pathway targets GlcNAc terminals to generate the functional glycans in various glycans. A recent study also suggested that β-1,3-N-acetylglucosaminyl-transferase is essential for functional glycosylation of α-DG in a prostate cancer cell line, supporting the view that GlcNAc terminals may be the target of Large-pathway[22].
Surprisingly, addition of GalNAc alone to the conditioned media of ldlD-LG cell culture remarkably enhances the Large-induced functional glycans on α-DG. In contrast, addition of both Gal and GalNAc to the ldlD-LG cell culture media resulted in producing much less functional glycans in the cells despite all glycosylation pathways are supposed to be restored in this condition. The phenomenon may be explained as follow. When the ldlD cells grown in the media without Gal/GalNAc, Large-pathway targets the GlcNAc terminals on O-linked and N-linked glycans to produce abundant functional glycans. Supplement of Gal alone to the culture media allows the cells to add Gal to the GlcNAc terminals on O- and N-linked glycans and results in inhibiting Large-pathway to target GlcNAc terminals to generate the functional glycans. While supplement of GalNAc alone to the cell culture the ldlD-LG cells add more GalNAc to the abundant Ser/Thr (up to 50) residues on the mucin domain to initiate the biosynthesis of mucin O-glycans. Sequentially, the GalNAc residues would be modified by endogenous βGlcNAc-transferases (βGn-Ts) to produce more GlcNAc terminals, since Gal is not available. Thus, Large modifies the abundant GlcNAc terminals to produce the most amount of the functional glycans among the cell culture conditions. Finally, while supplement of both Gal and GalNAc to the cell culture media, βGal-transferases (βGal-Ts) would modify GlcNAc terminals on all types of glycans, thus preventing Large to modify GlcNAc terminals to produced the functional glycans. Our data also suggested that fully restoration of mucin type of O-glycosylation with supplement of both Gal and GalNAc may compete with the functional O-mannosylation induced by Large. This is supported by recent report that the O-GalNAcylation and O-Mannosylation pathways are competing for the same sites of modification on α-DG[27]. Thus Large produced the least amount of the functional glycans among the cell culture conditions (Fig.5). The difference between the functional glycans biosynthesis in the different glycosylation conditions is unlikely due to protein degradation or other nonspecific effects caused by glycosylation defects. Since the functional glycans biosynthesis is suppressed in the better glycosylation conditions rather than in the worse conditions (Fig. 5).
To date, four O-glycans structures on α-DG have been reported: NeuAcα2-3Galβ1-4GlcNAcβ1-2Man (the tetra-glycan) from bovine brain and rabbit skeleton muscles [28], [29]. Galβ1-4(Fucα1-3)GlcNAcβ1-2Man (LewisX structure) from sheep brain [30], HSO3-3GlcAβ1-4GlcNAcβ1-2Man (HNK1 epitope) from rat brain [31], [32], and Galβ1-3GalNAc (Core-1) from myotubes [33]. The diversity of the O-glycan structures on α-DG allows α-DG to play distinct roles in various tissues to mediate the interactions with its variety of ligands. For example, HNK-1 epitope containing O-glycan mediates α-DG binding to laminins 10/11 in brain [32], while its core-1 structure of mucin-type glycan on α-DG mediates laminin-induced acetylcholine receptor clustering but not laminin binding activity in myotubes [33]. Our results suggested that the NeuAcα2-3Galβ1-4GlcNAcβ1-2Man glycan can not be the Large-induced functional glycans mediating laminin binding. Considering the N- and C-terminal domains of LARGE is related to bacterial α-glycosyltransferase and β-1,3-N-acetylglucosaminyl-transferase, respectively. In addition, Large itself is able to bind to α-DG. It is likely that Large function as glycosyltransferase modifying the GlcNAc to generate the functional glycans. Although the structure of Large-induced functional glycan remains to be determined, Our results nevertheless provided insights into the glycosylation of α-DG, which are illustrated in the Fig.6. 1) Large predominantly modifies O-mannosyl glycans to produce the functional glycans at stable overexpression levels in CHO cells, implicating that so does the endogenous Large. 2) Over expression of Large competes to modify GlcNAc terminals with galactosylation to generate the functional glycans on both O-linked and N-glycans, suggesting that suppressing galactosylation to produce more GlcNAc terminals on α-DG would significantly enhance Large function to produce the functional glycans, which may be a potential way to enhance the biosynthesis of the functional glycans on α-DG.
Mouse anti-glycosylated α-DG mAb IIH6 (Upstate Biotechnology, Inc.) was used at 1200 and 11000 dilutions for Immuno-fluorescent staining and Immuno-blotting assay, respectively. Anti-β-DG MAb 43DAG1/8D5 (hybridoma conditioned medium) from DHSB was used at 1200 dilution. Mouse anti-MYC mAb 9E10 (DHSB) was used at 1500 dilution. Rabbit anti-GM130 antibody (Stressgen) was used at 1500 dilution. Rabbit anti-laminin-1 polyclonal antibody (Sigma) was used at 1400 and 11500 dilution for Immuno-fluorescent staining and immuno-blotting assay, respectively. Anti-GFP mAb (JL-8) (Clontech) was used at 1100 for immuno-precipitation and 15000 for immuno-blotting assay. Laminins (laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, Sigma) was used at dilution 5 µg/ml for laminins overlay assay. Horseradish peroxidase-conjugated anti-mouse, anti-rabbit, and anti goat secondary antibodies (Sigma) were used at 15000–110,000 dilutions. Alexa-488, 594 conjugated secondary antibodies (Invitrogen) were used at 1200–1500 dilutions.
The mouse LARGE construct in pcDNA3.1/Myc-His(-)B vector was provided by Pamela Stanley [19]. Mouse DPM2 constructs in pCMV-Kan/Neo vector with or without GFP tag were purchased from Origene. The construct for human α-dystroglycan tagged with human Ig FC-fragment (HIG) was generated by cloning the a-DG cDNA fragment into the Pfuse-higG1-Fc vector (Invivogen). All constructs were confirmed by sequencing in Carolinas Medical Center molecular biology facility.
Pro5 and the glycosylation-deficient CHO mutants termed Lec1, Lec2, and Lec8 were purchased from ATCC [23]. The Lec15.2 and B421 cell lines were kindly provided by Mark Lehrman and Sharon Krag, respectively [34], [35]. These CHO cells were grown and maintained in F12 nutrition mix media with 10% fetal bovine serum (Invitrogen) in plates with 5% CO2 at 37°C, except that the Lec15 cells were grown at 34°C. For transfection, the cells were seeded in 6-wells plates, and were transfected using Fugene 6 reagents (Roche) followed the manufacturer's guidelines. For selecting stable cell lines expressing LARGE-MYC, the cells were cultured with G418 at 1.2 mg/ml (Invitrogen) 48 hours after transfection. The clones resistant to G418 were screened with laminin-dylight488. The positive clones binding to laminin-Dylight488 were picked and further confirmed with immuno-fluorescent staining with an anti-MYC monoclonal antibody.
Cell lysates were prepared from stable cell lines or transient transfectants 2 days after transfection. For each 10 cm plate, 1 ml of lysis buffer (1.5% Triton X-100 in PBS with protease inhibitor cocktail from ROCHE) was used to harvest the cells. The concentration of proteins in lysates was determined by Bio-Rad Dc protein assay. For electrophoresis, lysates (60 µg of protein) were boiled for 5 min in SDS-PAGE gels loading buffer. After electrophoresis in 4–12% gradient polyacrylamide gels (Invitrogen) at 168 V for 90–120 min, proteins were transferred to polyvinylidene difluoride membrane for 3 hours at 200 mA in transfer buffer containing 10% methanol. For immunoblotting, membranes were blocked in PBS with 10% fat free milk and incubated in the same solution with primary antibody at room temperature for 1 hour. After washing in PBS for 5X5 min, membrane was incubated in PBS/milk with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. After washing in PBS for 5X5 min, membrane was incubated in Super Signal West Pico™ chemiluminescence reagent (Pierce) and exposed to photographic film (Eastman Kodak Co.).
The cells were seeded in 96 well plates for overnight incubation. Then the cells were fixed and permeablized in 0.2% Triton X-100 in the laminin overlay buffer (LBB, 10 mM ethanolamine, 140 mM NaCl, 1 mM MgCl2 and 1 mM CaCl2, pH 7.4) for 8 min and blocked with 10% BSA for 1 hour at 37°C. Then 50 µl of 5 µg/ml laminins-DyLight-488 labeled in the LLB was incubated with the cells for 2 hours at 37°C. The plates were washed for 6 times with a micro-plate washer (BioTeck EXL405) and read with Infinite 500 microplate reader (TECAN). For staining the LARGE-MYC, an immuno-staining experiment was conducted with the anti-MYC polyclonal antibody.
The cells were seeded in 96 well plates for overnight incubation. Then the cells were incubated with 50 µl of 5 µg/ml laminins-DyLight-488 labeled in LBB for 6 hours at 37°C in cell culture incubator. The cells were washed with LLB twice and fixed. The cells were washed twice again and 50 µl LLB per well were added, and plates will be ready for microplate reading or capturing images with fluorescent microscopy.
The cell lysates were separated by SDS-PAGE gels and transfered to Nitrocellulose membranes. The membranes were blocked in 10% non-fat milk in LLB for 1 hour, and then incubated with 2.5 µg/ml laminin at 4°C overnight. After washing with LLB 5x5 min the laminin binding to α-DG was detected by standard Immunoblotting as describe above with a laminin polyclonal antibody (Sigma) followed by a goat anti-mouse IgG-HRP secondary antibody.
The cells were fixed, permeablized, and then blocked with 10% BSA in PBS for 1 hour. The primary antibodies in PBS with 10% BSA were incubated with the cells for 1 hour at room temperature (22°C) followed by addition of secondary antibodies conjugated with Alexa-488/594. After washing the cells with PBS for 2 times, the DAPI was used to stain the cells for 15 mins and wash 3 times with PBS. The images were captured with an inverted Olympus fluorescent microscope.
The deglycosylation procedure was followed the instruction by manufacture (NEB, Ipswich, MA). 50 µg of total protein extracted from indicated cells were boiled for 10 min in 1x Glycoprotein Denaturing Buffer followed by incubation with 500 units of PNGase F at 37°C for 4 hours. Samples were analyzed by immuno-blotting assay as described above.
We would like to thank to Mark Lehrman, Monty Krieger, and Sharon Krag for providing us the CHO mutant cells. We also want to express our appreciation to Pamela Stanley for providing us the mouse Large construct. We want to extend our appreciation to Hudson Freeze and Yiumo Chan for their critical reading of the manuscript and useful discussions.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work is supported by Carolinas Muscular Dystrophy foundation (http://www.givechf.org/index.php?option=com_content&task=view&id=28&Itemid=44). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Comments Thread For: Adrian Estrella-Fernando Saucedo Set For 12/6 in Mexico
Adrian Estrella continues the transformation from prospect to contender, as his team has agreed to terms for a showdown with recent super featherweight title challenger Fernando Saucedo.
The bout will take place on December 6 in Monterrey, Mexico, according to Estrellas promoter, Promociones del Pueblo.
Both fighters appeared in separate bouts on October 4, with their results going in opposite directions.
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I saw Estrella's fight on Televisa & he dominated a faded CC, but there were moments he looked very amateurish....Still, he's a walk ahead , hard punching guy who possesses more than enough 2 beat Saucedo...
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Estrella is garbage
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Anesthesiologist (pain control)
19 years of experience
Video profile
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44201 Dequindre Rd
Troy, MI 48085 Phone number & directions
44201 Dequindre Rd
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Gnutface is a gnutella client in the form of an eclipse plugin. See eclipse.org for information on the eclipse platform.
Gnutface uses the open source limewire engine for its gnutella engine. See limewire.org for information on limewire.
Gnutface has been released under the GNU GPL.
Note: The developers of this project do not condone copyright infringement or piracy and accept no responsibility for your misuse of this tool - use at your own risk.
Gnutface is currently in an alpha stage - most of the functionality is present but bugs are numerous. It should still be quite usable though. Note that Gnutface has been built agains Eclipse 3.0 M7. It has not been tested against other versions.
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Invisible Astro Healing Rhythm Quartet
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The Globalist
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We speak to the politician trying to ban outdoor smoking in Melbourne, check in with our man in Jerusalem, and ask whether an MBA is really worth the time and money.
31 January 2013
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There are no established genetic markers for prediction of outcomes after cyclophosphamide (CP)-containing adjuvant therapy for breast cancer. In an ancillary study to a Southwest Oncology Group trial (S8897), we investigated functional polymorphisms in 4 genes in CP pharmacokinetic pathways in relation to hematological toxicity and disease-free survival (DFS).
Germline DNA was available from 458 women who were at high risk of relapse and randomized to CAF vs CMF ± tamoxifen and from 874 women who had a presumed favorable prognosis and did not receive adjuvant therapy. Odds ratios for grade 3 and 4 hematological toxicity in the treated group and hazard ratios for DFS associated with selected functional polymorphisms in CYP2B6, CYP3A4, GSTA1, and GSTP1 were estimated by logistic regression and Cox proportional hazard regression.
Compared to women with AA genotypes, those with at least one GSTP1 variant G allele had reduced risk of grade 3 and 4 neutropenia (OR=0.63, 95% CI=0.41–0.97) and leucopenia (OR=0.59, 95% CI=0.39–0.89). No other associations between SNPs and toxicity or survival were found in the treated or untreated group.
Known genetic variants in genes involved in CP pharmacokinetics may not have major effects on DFS in breast cancer patients. The lower risk of developing high grade hematological toxicity among women with variant GSTP1 alleles suggests that genetic markers in combination with clinical factors may be useful in defining a subgroup of women who are less susceptible to adverse hematological toxicities with CP-containing therapies.
Adjuvant chemotherapy for early stage breast cancer patients has greatly improved both disease-free and overall survival by eliminating residual tumor cells after surgery (1, 2). However, due to lack of target specificity, systemically administered cytotoxic agents also cause damage to normal tissues, leading to severe and sometimes life-threatening toxicities (3). Treatment outcomes, including drug toxicities and disease-free survival (DFS), may vary greatly even among women receiving the same dose of the same drug. This is likely due, in part, to metabolic differences resulting in variability in effective drug dose delivery, as well as cell sensitivity to and defense against drug cytotoxicity.
Cyclophosphamide (CP) is a commonly used alkylating drug for breast cancer patients. There are large variations in pharmacokinetics of CP, with inter-individual variability estimated at 52% in the central volume of distribution, 36% in peripheral volume of distribution, and 22% in clearance of the active metabolites (4). The variation may be partially due to patient characteristics such as weight and age, but may also be due to genetic background (5). A number of genes including those encoding for Phase I activation enzymes and Phase II detoxification enzymes form complex pharmacokinetic networks which determine the effective dose delivered to target cells (6, 7). Those key genes are highly polymorphic among individuals, and functions of some common polymorphisms have been characterized, making them excellent candidates for pharmacogenetic studies.
CYP2B6 and CYP3A4 are two major hepatic enzymes that convert CP pro-drug to its active 4-hydroxylated metabolites. A neutral amino acid replacement in exon 4 of CYP2B6 resulting from a single nucleotide polymorphism (SNP) rs3745274 (Gln172His) is associated with increased catalytic activity in both cell cultures and human liver specimens (8, 9). For CYP3A4, SNP rs2740574 in the 5' promoter region is most commonly studied. Although it has little impact on constitutive expression (10, 11), it may result in lower enzymatic activity towards cyclophosphamide as shown recently in breast cancer patients (12). GSTA1 and GSTP1 are the two major detoxification enzymes for CP, conjugating reduced glutathione to cyclophosphamide metabolites. SNP rs3957356 in the 5' promoter of GSTA1 gene was reported to alter binding affinity of transcription factors and thus result in lower expression (13). A non-synonymous coding SNP rs977894 (Ile105Val) in exon 5 of GSTP1 decreases its glutathione conjugating affinity, resulting in compromised catalytic detoxification functions (14–16).
In SWOG S8897, a clinical trial with cyclophosphamide-containing regimens, we conducted an ancillary pharmacogenetic study to examine 4 common SNPs of CYP2B6, CYP3A4, GSTA1, and GSTP1 in relation to acute hematological toxicity and DFS after adjuvant chemotherapy.
This correlative study was nested in a completed clinical trial led by the SWOG for The Breast Cancer Intergroup (TBCI) of North America (S8897, INT0102). Details regarding trial design, patient characteristics, treatment plan, and outcomes were published previously (17–20). Briefly, women with stage T1 to T3a node-negative invasive breast cancer were eligible to participate. Based upon tumor prognostic characteristics including tumor size and hormone (estrogen and progesterone) receptor status, they were assigned to groups depending upon risk of recurrence (high risk, low risk, or indeterminate risk). Those in the indeterminate risk group were reassigned to either the high or low risk group based on S-phase fraction of their primary cancers as determined by flow cytometry. Women in the high risk group (either initially or after flow cytometry) were randomly assigned to an anthracycline-based regimen of 6 cycles of oral cyclophosphamide, intravenous doxorubicin, 5-fluorouracil (CAF) (21), or a non-anthracycline-containing regimen of 6 cycles of oral cyclophosphamide, intravenous methotrexate, and 5-fluorouracil (CMF) (22). After chemotherapy, patients in the treated group were subsequently randomized to tamoxifen for 5 years or not. Women in the low risk group served as an observational cohort and did not receive adjuvant chemotherapy or endocrine therapy. Prophylactic hematopoietic colony-stimulating factors (CSFs) were not administered at the time of the study.
Between 1989 and 1993, 3,965 eligible patients were registered onto the study. Of these, 2,690 patients were assigned to the high risk group (either initially or after flow cytometry) and received adjuvant therapy, and 1,206 were assigned to the low risk group and followed without adjuvant therapy, as shown in Figure 1. An additional 69 patients were not classifiable as either low or high risk and were excluded. Patient characteristics of age, race, and type of primary surgery were similar among all treatment groups. After a median follow-up time of 10.8 years, the study showed slightly better overall but not disease-free survival in the CAF arm than in the CMF arm, at an expense of higher toxicity (17).
For the present ancillary pharmacogenetic study, only women with archived, uninvolved lymph node tissue available for DNA extraction in the SWOG tissue bank were included. This group consisted of women assigned to the low risk group (initially and after flow cytometry, n=1,206) and women initially in the indeterminate risk group who were subsequently reassigned to the high risk group (n=527). Since all women with hormone-receptor negative breast cancer were classified as high risk initially, nodal tissue was not collected. Therefore, this analysis is restricted to women who were either estrogen receptor positive and/or progesterone receptor positive. The study also predates routine testing of HER2/neu status so HER2/neu status is also unknown. Written consent was obtained from patients for use of their tissues for ancillary research studies, and this study was approved by the Institutional Review Board at Roswell Park Cancer Institute. The number of specimens collected, and the number of patients for whom DNA was available, in each of the cohorts is included in Figure 1, according to REMARK criteria (23). These specimens have been used for previously reported studies of the association of germline variations of other genes and outcomes (18–20).
Two 5 micron paraffin-embedded formalin-fixed slides of normal lymph node tissue procured from the SWOG tissue bank in San Antonio were de-paraffinized and removed from slides. DNA extraction was performed with Qiagen DNeasy Tissue Kit (Qiagen Inc., Valencia, CA) as previously described (24), using a modified method based on Goelz et al (25). Adequate DNA for genotyping was obtained from 458 patients in the high risk group who received adjuvant therapy and 874 patients in the low risk group who were followed without adjuvant therapy (Figure 1).
Potentially functional SNPs in the 4 selected key genes involved in activation and detoxification of CP, including rs3745274 in CYP2B6, rs2740574 in CYP3A4, rs3957356 in GSTA1, and rs1695 in GSTP1, were genotyped by Sequenom matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Successful genotyping call rates varied between 88.4% and 99.6%, related to adequacy of DNA, with 100% concordant rates between 5% repetitive samples blindly included in the analyses.
Statistical analyses were prospectively planned in a formal, written protocol, and performed by the SWOG statistical center. For the analysis, we separated treated and untreated groups. Primary endpoints assessed in the study were high grade hematological toxicity and disease free survival (DFS). Per the protocol, DFS was defined as the time from the date of registration to the date of a second breast primary tumor, first recurrence, or death due to any cause, but not a new non-breast primary cancer (26). S8897 was performed prior to development of the NCI Common Toxicity scales, and therefore standard SWOG toxicity criteria applied per protocol at the time were used (27). In brief, grade 2–4 neutropenia was defined by count of neutrophils between 1,000–1,500/mm3, 500-1,000/mm3, and <500/mm3, respectively; and grade 2–4 leucopenia was defined by count of leukocytes between 2,000–3,000/mm3, 1,000–2,000/mm3, and <1,000/mm3, respectively. The highest grade toxicity occurred during the treatment was recorded and used in this analysis. Prevalence of acute toxicities within 1 year of registration by treatment group in the trial has been previously reported (17), and was similar among the subset of patients included in this ancillary study. Hematological toxicities, mainly neutropenia and leucopenia, were the most common acute toxicities in all treatment groups; grade 4 non-hematological toxicity, including severe cardiac toxicity, was rare (n=2/458). Thus, primary endpoints in toxicity analysis were grades 3 and 4 neutropenia, grades 3 and 4 leucopenia, and combined grade 4 hematological toxicity.
For toxicity analysis in women who received adjuvant therapy, Chi-square test or Fisher's exact test was first performed to examine genotype distribution by toxicity grade. Considering the incomplete knowledge of exact functions of the genes and the genotypes, if homozygous and heterozygous genotypes had similar frequency distributions by toxicity grade, the categories were combined under an assumption of dominant or recessive genetic models. Similarly, rare homozygous genotypes were collapsed with heterozygous genotypes for power considerations. Unconditional logistic regression was used to estimate associations between each SNP and risk of high grade toxicity. For neutropenia and leucopenia, grade 3 and 4 toxicities were first combined and tested together and then tested in an ordinal model under an assumption that there is a linear relationship between toxicity grades. In addition to a binary outcome of grade 4 hematological toxicity (yes vs no), discrete count of grade 4 toxicity based on neutropenia and leucopenia (0, 1, and 2) was also used as an ordinal outcome in logistic regression models. Unadjusted odds ratios (ORs) and 95% confidence intervals (CIs) were first derived, and then were adjusted for age, menopausal status, chemotherapy arms (CAF vs CMF), tamoxifen treatment, and time from surgery to randomization. Additional control for race or excluding non-white race from the analyses did not substantially change the results. According to the pre-planned analytic protocol, for a SNP with a minor allele frequency in the range of 0.05 to 0.50, we would have at least 80% power to detect a minimum odds ratio in the range of 0.56–0.64 for grade 4 toxicity. Four SNPs were tested in the analyses; and for each SNP, two genetic models including dominant and ordinal models and three toxicity outcomes including neutropenia, leucopenia and grade 4 hematological toxicity were tested, yielding a total of 24 tests. To address the concern of false positivity due to multiple testing, we calculated the false positive report possibility (FPRP) using the method by Wacholder et al (28), assuming a prior probability for associations with toxicities of 0.1, 80% statistical power, and a FPRP below 0.5 for suggestive associations and a FPRP below 0.2 for more definitive associations.
Potential modifying effects of genotypes on DFS were examined by contrasting event-free time between those carrying variant alleles and those with homozygous common alleles. Kaplan-Meier survival curves were generated and the differences were tested by log-rank test. To control for potential confounders, Cox proportion hazard models were used to adjust for age, menopausal status, and for the treated group only, also time from surgery to randomization, chemotherapy arms (CAF vs CMF), and tamoxifen treatment.
To test interacting effects from chemotherapy regimens and tamoxifen treatment, we fitted in the regression models a productive term between treatment regimen and genotypes. The significance of the interactions was assessed by likelihood ratio test. To test synergistic and/or additive effects among multiple SNPs, the number of "at-risk" alleles that increases CP activation or decreases detoxification was tallied (range 0–8) and categorized into ranks. Toxicities and DFS were then compared across those ranks.
Characteristics of patients included in this ancillary study are summarized in Table 1; there were no differences between this subset of patients and those in the entire trial (17). As expected, women in the treated group tended to be younger, premenopausal, and to have larger tumors than those in the untreated group, who were at lower risk of recurrence. During a median 10.8 years of follow-up, there were 129 events in the treated group and 257 in the untreated group. S8897 was conducted prior to the era of CSF support. Of the patients who received adjuvant therapy, there were few reports of thrombocytopenia (2%), but 67% had grade 3 and 4 neutropenia and 62% had grade 3 and 4 leucopenia. Thirty-five percent (35%) of patients had one grade 4 hematological toxicity, and 12% experienced two grade 4 events. Genotype frequencies are summarized in Table 2, and were similar to those in the SNP500Cancer database.
As shown in Table 3, there were no significant associations between the polymorphisms in CYP2B6, CYP3A4 or GSTA1 and risk of grade 4 hematological toxicity, or with risk of high grade neutropenia or leucopenia (data not shown). However, there were notable associations between the GSTP1 polymorphism and reduced risk of high grade hematological toxicity, with similar ORs in unadjusted and adjusted models (Table 4). Compared to women homozygous for the common allele, those carrying the less active variant G allele had significantly lower risk of grade 3 and 4 neutropenia (adjusted OR=0.63, 95% CI=0.41–0.97, p=0.04) and grade 3 and 4 leucopenia (adjusted OR=0.59, 95% CI=0.39–0.89, p=0.01). Associations with risk of any grade 4 hematological toxicity were of borderline significance (adjusted OR=0.72, 95% CI=0.48–1.07, p=0.11). Results from the models with the ordinal outcomes (grade ≤2, 3 and 4; or count of grade 4 of hematological toxicity 0, 1 and 2) were consistent with the above models with binary outcomes. FPRP calculation indicates that the strength of the observed associations between the GSTP1 polymorphism and hematological toxicity was between suggestive (FPRP <0.5) to more definitive (FPRP <0.2).
Associations between each individual SNP and DFS are shown in Table 5. There was no statistically significant association of any SNP in any of the evaluated genes with DFS in women receiving or not adjuvant therapy. No significant interactions between genotypes and treatment randomization (CMF vs CAF, tamoxifen yes vs no). In analyses to examine synergistic and/or additive effects of the four selected SNPs, there were also no associations of hematological toxicities or DFS with total number of at risk alleles (data not shown).
In this pharmacogenetic study, there were significant associations between a common genetic polymorphism in GSTP1 and acute hematological toxicity, but no associations with DFS. Selected polymorphisms in 3 other key genes involved in CP metabolism (CYP2B6, CYP3A4, and GSTA1) were not associated with hematological toxicity or DFS following adjuvant therapy. Because CSFs for neutropenia prophylaxis are now commonly used in patients receiving breast cancer adjuvant chemotherapy, our findings of significant associations between the GSTP1 polymorphism and hematological toxicity suggest that a subgroup of women may be at low risk. Thus, CSF support may not be necessary when considering the cost and potential side effects from growth factors, including bone pain and decreased bone mineral density. However, further confirmation in other datasets using archived specimens, or in a prospective trial, from patients who received cyclophosphamide without CSF support is required before such a strategy would be recommended
The variant G allele of GSTP1 polymorphism has been associated with increased risk of toxicity in colorectal cancer patients (29) and increased risk of neutropenia in lupus erythematosus patients treated with CP (30). However, in women with breast cancer, this SNP was not associated with adverse drug reactions after CP-containing chemotherapy in two previous studies (31, 32). In a small group of 94 women receiving 6 cycles of anthracycline-based CEF regimen for breast cancer, GG genotypes of GSTP1 were associated with increased risk of grade 3 and 4 hematological toxicity (OR=6.4, 95% CI=1.05–39.0) (33). Surprisingly, we found in our study an inverse association, which cannot be explained by direct drug detoxification function of GSTP1. However, there are other reports in the literature showing similar trends. Compared to GG genotypes, the more active AA genotype has been associated with increased risk of oxaliplatin-induced neuropathy (34), docetaxel-induced neuropathy (35), and cisplatin-induced hearing impairment (36). A possible alternative mechanism to explain these unexpected findings in drug-related toxicity is through the novel roles of GSTP1 in cellular stress response signaling as an inhibitor of c-Jun N terminal kinase (JNK) (37), a key cellular stress mediator which can activate downstream transcription factors to up-regulate a number of antioxidant cellular response genes. It was speculated that less active G alleles result in increased JNK activity and elevated expression of downstream cellular stress defense genes which may protect cells from drug cytotoxicity (34). Slower consumption of reduced glutathione by the less active GSTP1 variant may also facilitate the defense against cellular stress and may prevent cell apoptosis (38). In addition, it is possible that the functional alteration of the G allele may be drug-specific, as supported by findings in cultured cells that the "less active" GG genotype showed enhanced cisplatin detoxification compared to the AA genotype (39). Our study, along with others, calls for further drug- and tissue-specific functional characterization of this SNP.
Despite significant associations with reduced risk of toxicity, GG genotypes of GSTP1 were not associated with poorer DFS (HR=1.19, 95% CI=0.66–2.13) among women receiving chemotherapy (Table 5). The lack of concordance between survival and toxicity may be due to differential capacities of normal and malignant cells in dealing with drug cytotoxicity, which may be further attributed to somatic changes incurred during tumorigenesis in cancer cells. The above null result, however, is unexpected based on our previous findings in a smaller study, where the HR for overall breast cancer survival was 0.3 (95% CI=0.1–1.0) for GG genotypes compared to AA genotypes (40). Similar results were also reported in a study with Chinese breast cancer patients (41). Heterogeneity in patient characteristics and treatment regimens among those studies may partially account for the inconsistent findings.
There were no significant associations between treatment outcomes and polymorphisms in CYP2B6, CYP3A4, or GSTA1 genes. The null findings in our study could be due to redundancy in drug metabolism genes. Although we selected four key genes in CP metabolism pathway, a number of other genes in the same pathway may neutralize any functional changes due to the selected genetic variations.
In conclusion, in a cooperative group clinical trial for breast cancer, we found that genetic polymorphisms in key genes in cyclophosphamide metabolism were not associated with DFS following adjuvant chemotherapy. Of the genes evaluated, only GSTP1 polymorphism was significantly associated with risk of acute hematological toxicity. Our results indicate that genetic variations in cyclophosphamide pharmacokinetic pathways may not be candidate markers for predicting treatment outcomes. Women carrying GSTP1 variants may be at lower risk of neutropenia, suggesting that genetic markers in combination with other clinical and molecular factors may be useful in predicting risk of neutropenia, and for those at low risk, prophylactic CSF may not be necessary. These results need to be further validated in independent studies before clinical recommendations can be made.
In the context of a clinical trial of cyclophosphamide-containing chemotherapy for breast cancer, there were significant associations between germline GSTP1 variants and reduced risk of acute neutropenia and leucopenia after treatment. As a prophylactic measure for febrile neutropenia and infection, hematopoietic colony-stimulating factor (CSF) is commonly administered during standard chemotherapy; however, for some patients, the cost-effectiveness may not be well justified. Side effects including bone loss and bone pain may also arise from CSF use. Our findings on GSTP1 indicate that genetic variations may contribute to chemotherapy-induced neutropenia. If validated, integration of genetic factors with clinical and molecular characteristics could be used for a composite algorithm to predict risk of neutropenia prior to treatment, to facilitate clinical decision making on regimen and dosing selection of chemotherapy, as well as CSF support.
Grant support:
This investigation was supported in part by the following R01 and PHS Cooperative Agreement grant numbers awarded by the National Cancer Institute, DHHS: CA095222, CA32102, CA38926, CA02599, CA13612, CA22433, CA27057, CA37981, CA46282, CA20319, CA35431, CA76447, CA45560, CA12644, CA14028, CA58416, CA04919, CA35090, CA35176, CA58686, CA58861, CA46113, CA58882, CA35128, CA74647, CA46136, CA45450, CA35261, CA35192, CA12213, CA16385, CA58658, CA46441, CA58723, CA45377, CA35119, CA42777, CA73590, CA114558-02, CA35178 and CA35262. Drs. Ambrosone, Hayes, Hortobagyi and Rae are recipients of funding from the Breast Cancer Research Foundation. Dr. Yao was partially supported by a Department of Defense award DAMD W81XWH-08-1-0223.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Belly up to the kombucha bar
Fermented tea aims to compete with soft drinks more than beer
Ian Anderson 5 p.m., Sept. 30
Posted September 13, 2011
Beauty and the blight on Mission Bay.
Cityzella terrorizes humble trailer folk at De Anza Cove.
This was a blast from the past for me. I lived in that trailer park, until I was about 4yrs old. I think it's nice to think of something from my childhood is still there. I haven't seen it in years, it's sad to think of it as an eye soar. Maybe we need to get back to simpler times, like trailer parks. Pacific Beach was the best place to grow up. Maybe someone should help the residents clean it up :)


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Bloomington Buffet Restaurants
1. Michael's RestaurantMenu
Michael's has a wonderful Sunday Brunch!! \r \r My family and I attend every Sunday after Church. They have such a large variety of foods to cho…
2. Pizza RanchMenu
1211 Holiday DrBloomington, IL 61704
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come hungry leave happy. I will be coming back when ever I can.
6. China Wok
We used to like this place but for the meal we just had no flavor, husband ordered spicy it was not. plus we were to have egg rolls we did not. I th…
7. Lin's BuffetMenu
756 W Jackson St, Morton IL, 61550Morton, IL 61550
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Showing1-7 of 7results


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Primary Care Doctor, General Practitioner, Internist
6 years of experience
6 years of experience
Georgia State University
80 Jesse Hill Jr Dr SE
Atlanta, GA 30303 Phone number & directions
80 Jesse Hill Jr Dr SE
Atlanta, GA 30303 Phone number & directions
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Khirbat Zalafa
|Khirbat Zalafa
|خربة زلفة
|Name meaning
|"The cistern"[1]
|Also spelled
|Khirbet Zalafa, Zalafa
|32°24′9.10″N 34°56′43.96″E / 32.4025278°N 34.9455444°ECoordinates: 32°24′9.10″N 34°56′43.96″E / 32.4025278°N 34.9455444°E
|Palestine grid
|210[2] (1945)
|7,713 (6,865 Arab, 617 Jewish, 231 Public) dunams
7.7 km²
|Date of depopulation
|April 15, 1948[3]
|Cause(s) of depopulation
|Fear of being caught up in the fighting
Khirbat Zalafa (Arabic: خربة زلفة) was a small Palestinian Arab village in the Tulkarm Subdistrict, located about 15 kilometers (9.3 mi) northwest of Tulkarm. It was depopulated during the 1948 Palestine war. It was captured by Yishuv forces on April 15, 1948 as a part of operation "Coastal Clearing."[4]
Originally, the people of Khirbat Zalafa came from Attil to farm the village land. Gradually they settled in the village so they could be closer to their land. In the late 19th century, Khirbat Zalafa was described as a small hamlet with springs to the south.[5]
In the 1922 census of Palestine there were 63 villagers, all Muslim.[6] At the time of the 1931 census, the village was counted under Attil, together with Jalama and Al-Manshiyya.[7]
The village had a small core of houses, with many dwellings scattered throughout on the agricultural lands. The agriculture was based on watermelons, vegetables, grain and olives. In 1944/45 6,798 dunums were allotted to cereals, 38 dunum was devoted to citrus and bananas ad 6 dunums were irrigated or used for orchards.[8] while 3 dunams were classified as built-up, urban land.[9]
1948 and after[edit]
The Palestinian historian Walid Khalidi described the village in 1992: "The village has been completely levelled. Both the original site and the surrounding lands are covered with Israeli citrus orchards."[10]
There are no Israeli settlements on village land.[10]
See also[edit]
- ^ Palmer, 1881, p. 196
- ^ Department of Statistics, 1945, p. 21
- ^ Morris, 2004, p. xviii, village #185. Also gives cause(s) of depopulation.
- ^ Morris, 2004, p. xviii, village #185
- ^ Conder and Kitchener, 1882, SWP II, p. 153. Quoted in Khalidi, 1992, p. 568
- ^ Barron, 1923, Table IX, Sub-district of Tulkarem, p. 28
- ^ Mills, 1932, p. 53
- ^ Government of Palestine, Department of Statistics. Village Statistics, April, 1945. Quoted in Hadawi, 1970, p. 126 Also Khalidi, 1992, p. 568
- ^ Government of Palestine, Department of Statistics. Village Statistics, April, 1945. Quoted in Hadawi, 1970, p. 176
- ^ a b Khalidi, 1992, p. 568
- Barron, J. B., ed. (1923). Palestine: Report and General Abstracts of the Census of 1922. Government of Palestine.
- Conder, Claude Reignier; Kitchener, H. H. (1882). The Survey of Western Palestine: Memoirs of the Topography, Orography, Hydrography, and Archaeology. 2. London: Committee of the Palestine Exploration Fund.
- Department of Statistics (1945). Village Statistics, April, 1945. Government of Palestine.
- Hadawi, Sami (1970). Village Statistics of 1945: A Classification of Land and Area ownership in Palestine. Palestine Liberation Organization Research Center.
- Khalidi, Walid (1992). All That Remains: The Palestinian Villages Occupied and Depopulated by Israel in 1948. Washington D.C.: Institute for Palestine Studies. ISBN 0-88728-224-5.
- Mills, E., ed. (1932). Census of Palestine 1931. Population of Villages, Towns and Administrative Areas. Jerusalem: Government of Palestine.
- Morris, Benny (2004). The Birth of the Palestinian Refugee Problem Revisited. Cambridge University Press. ISBN 978-0-521-00967-6.
- Palmer, E. H. (1881). The Survey of Western Palestine: Arabic and English Name Lists Collected During the Survey by Lieutenants Conder and Kitchener, R. E. Transliterated and Explained by E.H. Palmer. Committee of the Palestine Exploration Fund.
External links and references[edit]
- Welcome To Zalafa, Khirbat
- Zalafa, Khirbat, from the Khalil Sakakini Cultural Center
- Survey of Western Palestine, Map 11: IAA, Wikimedia commons


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Upcoming Events
SFD Safety Committee Meeting at Co 52 Sept 7, 2016 1830 Hours
Welcome Back Shippensburg University Students
Sun, Aug 28th, 2016 8:32:00 pm - Members of SFD welcome back to our community the Students of Shippensburg University. We look forward meeting you and hopefully encourage many of you to volunteer at one of our three fire station.
Shippensburg Corn Festival
Sun, Aug 28th, 2016 8:28:00 pm - Shippensburg Corn Festival keeps area first responders busy to ensure the safety of the festival. Members enjoy the time to interact with the community as well.
Prince Street Church Youth Group
Wed, Aug 24th, 2016 8:24:00 pm - The Youth Group of the Prince Street Church "United Brethren in Christ Church recognized our local volunteers at their August 24th, 2016 meeting. We had the opputunity to talk to them on the importance of volunteering in your community.
Fire Extinguisher Training at SU
Fri, Aug 19th, 2016 8:21:00 am - Members provided Fire Extinguisher to the RA's at Shippensburg University. During this time members reviewed proper emergency procedures that should be observed while acting as RA's. Personnel also provided fire extinguisher training to the Shippensburg Area School District.
Mass Causality Drill
Tue, Aug 16th, 2016 9:48:00 pm - The Shippensburg Fire Department with Shippensburg Area EMS and several other agencies participated in a mass causality drill held at the Southampton Township maintenance building. This drill was intended to give first responders and command staff personnel the opportunity to preform a hands on drill to simulate a mass causality incident. This gives everyone the opportunity to sharpen skills and make changes to guidelines if
Ship Cares
Sun, Aug 14th, 2016 9:51:00 pm - Thanks to a group of community members that came together and support and honor local first responders. This was a great evening to connect with our community members.
Active Shooter Tabletop
Thu, Aug 11th, 2016 9:40:00 pm - On August 11, 2016 members of the emergency services along with the Shippensburg University,Shippensburg Area School District and the Cumberland County Department of Public Safety participated with an Active Shooter Exercise at Grace B Luhrs Elementary. The Tabletop was managed by MG Tactical. This took months of planning and cooperation from multiple agencies. Thanks to all that participated.
Fire Safety Event
Tue, Aug 9th, 2016 8:33:00 pm - On Tuesday August 9th. 2016 members spent the evening at the Oasis of Love Vacation Bible School honoring local heros. Members had a great time teaching the children Fire Safety and interacting with the children and staff.
Local Hero Night
Wed, Jul 27th, 2016 5:06:00 pm - All local First Responders and community members are encouraged to attend.
Jr Fire Academy Granduation
Sun, Jul 24th, 2016 7:07:00 pm - Congratulations to the Junior Members of the Vigilant Hose Company and CV Hose Company for Graduating from the Franklin County Junior Fire Academy.
© Shippensburg Fire


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1999 Chevrolet Suburban 2500 Repair and Maintenance
A guide to problems, costs, maintenance and repair for your 1999 Chevrolet Suburban 2500
Verified by mechanics, reported by drivers like you
Ask a mechanic in our community
Never overpay. Check prices with our estimator
1999 Chevrolet Suburban 2500 ProblemsEngine Misfire Due to Worn Valve Seat
The engine may develop a misfire due to worn valve seats. This fault will cause the Check Engine Light to illuminate. Our technicians tell us the misfire may or may not be felt while driving. They further recommend that both cylinder heads be removed and thoroughly inspected and repaired as necessary.
Illumination of the Check Engine Light can be caused by a loose or worn gas cap.
One or more fuel injectors may become stuck closed causing an engine misfire and the Check Engine Light to illuminate. Our technicians tell us that this problem can be caused by some fuel additives. There are special procedures which may be able to restore the affected injector. If this fails the affected injector must be replaced.
1999 Chevrolet Suburban 2500 QuestionsWhy Does AC Compressor Clutch Engage and Disengage (2 answers)
I turn it on and it turns on then off in like 30 seconds
solo lo hace cuando llueve y la camioneta se moja al otro dia no arranca y si llueve todo el dia pues no arranca mientras no se moje funciona normalmente pasan dias o meses sin llover y la camioneta no falla espero pudieran orientarme que hacer gracias.
I am leaking power steering fluid. I have replaced both lines. I think it is coming from the joint between the hydroboost and brake booster. Is there a seal I can replace or do I have to replace the whole booster? Thanks!


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The episode taken from THIS SITE
Two tops Nick Moretti and Tyler Saint go through a series of rounds which are set to decide who will be the bottom in the final sex round. There is three eight minutes rounds in each Naked Kombat match, which include plenty of muscle slipping, cock sliding and ass slapping action, this time with water gushing form above on both fighters. They battle hard, but there is only one winner who is allowed to have a way with his opponent's ass. Watch inside who will that be?