text:
[kwin-i-deen, -din] /ˈkwɪn ɪˌdin, -dɪn/
noun, Pharmacology.
a colorless, crystalline alkaloid, C 2 0 H 2 4 N 2 O 2 , isomeric with , obtained from the bark of certain species of cinchona trees or shrubs, used chiefly to regulate heart rhythm and to treat malaria.
a crystalline alkaloid drug that is an optically active diastereoisomer of quinine: used to treat heart arrhythmias. Formula: C20H24N2O2
quinidine quin·i·dine (kwĭn'ĭ-dēn')
A colorless crystalline alkaloid that is a stereoisomer of quinine and is used as a treatment for malaria and cardiac arrhythmias.
Tagged: q
Read Also:
- Quiniela
[keen-yel-uh; Spanish kee-nye-lah] /kinˈyɛl ə; Spanish kiˈnyɛ lɑ/ noun 1. .
- Quinine
[kwahy-nahyn, kwin-ahyn or, esp. British, kwi-neen] /ˈkwaɪ naɪn, ˈkwɪn aɪn or, esp. British, kwɪˈnin/ noun, Chemistry, Pharmacology. 1. a white, bitter, slightly water-soluble alkaloid, C 2 0 H 2 4 N 2 O 2 , having needlelike crystals, obtained from cinchona bark: used in medicine chiefly in the treatment of resistant forms of malaria. 2. […]
- Quinine-water
noun 1. carbonated water containing lemon, lime, sweetener, and quinine, often used as a mixer.
- Quinn
/kwɪn/ noun 1. Anthony. 1915–2001, US film actor, born in Mexico: noted esp for his performances in La Strada (1954) and Zorba the Greek (1964)


text:
- 59 years old
- Date of birth: Jun 20, 1955
- Date of passing: Dec 28, 2014
|Let the memory of leanne be with us forever
"I miss you mom and I love you so much. I can't wait to see you soon!!!!! You are in my heart forever and always. Thanks for being the best mom. I wish your here with me. Ps. RIP MOM!!!! <3"
"I love you mom and I miss you so much. You be in heart forever and always. your the best mom that any one could have. I wish it's was me who died not you. Ever since you been gone I been hurting deep down inside. My life emotions are very confuse and all over the place and sometimes I think I can't be happy ever agian. I know we had differences but you still ended up loving me and forgiving for the pain I cause you and I really sorry for all the horrible shit I did. I feel bad for things I said and now your gone its hurt me worst then before. I know i can be a horrible bipolar bitch some times but I am thankful for the life you gave me. I happy to have you as my adopted mom. I write song to tell you how much you mean to me mom.I love you and i really miss. I can't express my emotions and thoughts until I can feel my happy and myself again. We all wish we can be a family again living in our house.
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text:
Dallas Cowboys receiver Dez Bryant isn't about to provide any sound bytes leading into his matchup with Seattle Seahawks cornerback Richard Sherman on Sunday.
Bryant and Sherman are reportedly friendly acquaintances after meeting at the ESPYs last summer, and there's also a clear respect there. Not that Bryant has ever been a big smack talker, off the field anyway, but he had nothing but subdued praise for the effusive Seahawks star this week.
"It can't be personal. You've got to go out there and focus on what the offense is doing and what the play-calling is doing," Bryant said, per Jon Machota of the Dallas Morning News. "You've got to go out there and focus. I'm just looking at it as he's an outstanding football player and you've got to be prepared when you go against someone like that."
Of course Bryant, who has 32 catches for 376 yards and four touchdowns on the year thus far, knows their relationship could very well become a lot less friendly and a lot less quiet after kickoff.
"That's just the nature of the game," Bryant said. "I'm pretty sure he's going to say something to me. I'm pretty sure I'm going to say something to him. But at the end of the day, when it's all said and done, we're going to congratulate one another and talk and take pictures and get back on the plane and go our separate ways."
Stay on top of the latest Dallas Cowboys news with NBCDFW.com. Follow NBC DFW on Facebook or have breaking Cowboys news sent directly to your phone with Blue Star SMS alerts. Text "bluestar" to 622339 (NBCDFW) to subscribe, text HELP for more info, and text STOP to end the subscription. Message and data rates may apply.Read more about it here.


text:
8/35b Halls Head Parade Halls Head 6210 WA
This property has been relisted
8/35b Halls Head Parade,Halls Head WA
House For Rent
$250 per week
Lovely 2 bedroom, 1 bathroom low maintenance unit in a beach side location. Modern wooden floor boards, open plan living area, reverse cycle air-conditioning with ceiling fans to bedrooms. There is a heated, outdoor shower for after a swim at the beach. Enjoy sitting out the back in the courtyard. You can walk across the road to the beautiful Halls Head beach or stroll down to the Dome for a coffee and cake. Enjoy this beach lifestyle. Store room not for tenants use however there is a single enclosed lockable electric carport with built in shelves for storage space. The property also features crim safe security at the rear. Low maintenance gardens, pets by approval. Available Soon. PLEASE NOTE: Furnishings are not included
Property Code: 474
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Real Estate For Rent in Halls Head, WA 6210 priced between $180 and $330


text:
Here is a collection of my first 12 art lessons I created for my Recycled Art Journal Project.
The home made journals are created from:
• 6″ x 9″ rectangles cut from old boxes (Cereal boxes recommended)
• 9″ x 12″ art paper cut in half
• Book rings
• 2-hole punch from Staples
• Six Art Elements lessons: Line, Shape/Form, Space, Color, Value and Texture
• Six lessons inspired by famous artists: Delaunay, Robert Indiana, Matisse, Magritte, Mondrian, and Picasso
FINISHED ART DIMENSIONS: 6″ x 9″
PURCHASE AND DOWNLOAD: Follow the Paypal screen prompts to select your payment options. Upon completion, use the "Return to RQS Productions, Inc." link to open your Checkout page containing your PDF download link. The PDF file will also be automatically sent to the email address listed on your Paypal account.
GUARANTEE: If your purchase is lost, deleted or damaged, or for any reason, I'll email the PDF file, on request. KB


text:
Practical Handbook of Microbiology, 3rd Edition (2015 Release)
Practical Handbook of Microbiology, 3rd Edition (2015 Release)
by Emanuel Goldman (Editor), Lorrence H Green (Editor)
The Practical Handbook of Microbiology presents basic knowledge about working with microorganisms in a clear and concise form. It also provides in-depth information on important aspects of the field?from classical microbiology to genomics?in one easily accessible volume.
This new edition retains the easy-to-use format of previous editions, with a logical presentation of frequently used reference data that enables readers to rapidly locate the information needed.
New chapters have been included in this edition, including a noteworthy one on the business aspects of microbiology that has been added to address the needs of investors looking to understand the science behind companies that they are contemplating funding and scientists that are interested in commercializing their research. In addition, chapters have been added on new microorganism-based disease and pathogenic mechanisms.
All chapters from the previous edition have been revised and updated. Major topics covered include almost all studied bacteria, and introductions to fungi, parasites, and viruses, as well as methods of culture collection, enumeration, and preservation of microorganisms, diagnostic medical microbiology, mechanisms of antimicrobial agents, and antibiotics and antifungal agents.
Although this book will be of use to anyone interested in the subject matter, it will be of particular benefit to specialized microbiologists as well as those who simply use microbiology as an adjunct to their own discipline, in finding relevant information quickly and easily.
Download ebook Practical Handbook of Microbiology, 3rd Edition (2015 Release) pdf
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text:
Ideologies and Technologies of Motherhood charts new territory by exploring the notion of motherhood for women of differing classes, races, religions and nations in the light of various strategies and new technologies used to attain motherhood. This collection of essays by leading experts offers a wealth of ethnographic case studies that illustrate how women experience and practice motherhood in the United States, Egypt, Israel, South Africa, the United Kingdom, and other nations.
By examining such topics as transracial adoption, surrogate motherhood, in vitro fertilization, pregnancy loss, and caring for disabled children, the contributors reveal how race, gender, kinship and personal identity are transformed through the lens of motherhood, and how motherhood itself is being reconfigured across national and cultural contexts.


text:
Johnny Marr talks about his 1962 Epiphone Coronet on [this page](http://www.johnny-marr.com/guitarchestra-2/1962-epiphone-coronet) of his website. He says, "This is a 1962 Epiphone Coronet. Shortly after I got it I put it in "Nashville Tuning", which means putting on an electric 12 string set (the bottom four strings are an octave higher than standard). It feels like your playing backwards because the higher strings are at the bottom. I used it to double a lot of the Rickenbacker arpeggios on Smiths records, most notably on "William It Was Really Nothing", it's also the main guitar on "Half A Person"."more
Ronnie Wood uses this guitar when was touring with Rod Stewartmore
Ryan has mostly being seen with this guitar during his side project exclamation ponymore
Paul Stanley is seen with an Epiphone Coronet in this backstage photo from some time around late 1975.more
Ace Frehley "I used to play an Epiphone when I was about 18, 19, kind of like the Steve Marriott model he used in Humble Pie."more
In this photo, Walla is seen playing Epiphone Coronet.more
Tegan is playing an Epiphone Coronet in the studio in this photo.more
Pete Doherty can be seen playing a vintage cherry red finish Epiphone Coronet Electric Guitar in this photo. He has played it with several of his bands, including Libertines and Babyshambles. It most closely matches a 1960s model, with a single P-90 pickup in the bridge position. There was a brief run of Korean-made 1990s Coronets which have a single coil pickup in the neck position, thus excluding that as a possibility for the one Pete Doherty plays. In terms of the headstock, the original and 90s reissued Coronets varied in terms of headstock style - some had six tuners all on one side, while others had 3-per-side Gibson-style headstocks. Doherty's particular Coronet has the former.more
Luke was playing this suburst Epiphone Coronet when he covered with Mark Foster Alabama Shake's song "Hold On" during a Foster The People gig That was the only occasion of seeing him playing that guitar You can find the video here: https://www.youtube.com/watch?v=MyZDkW9vg7Mmore
Neil can be seen playing an Epiphone Coronet in this video.more
Ricky's playing a Epiphone Coronet on "6060-842".more
With an Equipboard account you can rate this item, add it to your collection, submit a review to discuss what you like and dislike about it, and associate Epiphone Coronet to artists that use it. Create an account!


text:
+914 Strength
+1456 Strength
+596 Strength
+739 Strength
+475 Strength
+1171 Strength
+474 Strength
+705 Strength
+726 Strength
+553 Strength
+450 Strength
+749 Strength
This feature lists your most prevalent gear bonuses. You may select a bonus to see which items provide that bonus.
Strength: 10375
Primary attribute for Barbarians and Crusaders.
Increases all damage for Barbarians and Crusaders by 10375.00%.
Increases Armor by 10375.
Dexterity: 77
Primary attribute for Demon Hunters and Monks.
Increases all damage for Demon Hunters and Monks by 77.00%.
Increases Armor by 7.70%.
Intelligence: 77
Primary attribute for Witch Doctors, Wizards and Necromancers.
Increases all damage for Witch Doctors, Wizards and Necromancers by 77.00%.
Increases resistances by 7.7.
Vitality: 4860
Increases Life, allowing more damage to be taken before dying.
Each point of Vitality increases Life by 100.
Armor: 15389
Reduces all damage from enemies of the same level as you by 0.00%.
Damage: 1314900.00
The amount of damage per second you can deal.
Damage is based on your weapons, attributes, attack speed, Critical Hit Chance,
Critical Hit Damage, passive skills, and dual-wielding attack speed increase.
Toughness: 12799700
The total amount of raw damage you can take.
Toughness is based on your maximum Life, armor, dodge chance,
average resistances, and missile, melee, and elite damage reduction.
Recovery: 567367
The amount of Toughness you can recover every second in combat.
Recovery is based on your Toughness, Life per Hit, Life per Second, Life per Kill,
Life per Fury/Spirit/Wrath Spent, Life Steal, and Health Globe Healing Bonus.
Maximum Life: 486316
If you lose all your Life, you die.
Life can be increased by skills, items, and Vitality.
Fury 132/132
Generated when the Barbarian uses Fury generating skills
or takes damage. Used for more powerful Barbarian attacks.
|Crippling Shot
|Dirty Fighting
|Powered Shot
|Powered Armor
|Focused Mind
Last updated on Mar 26, 2017 2:48 PM PDT


text:
Saturday, November 19 / December 2, 2006: Gal 1:3-10, Luke 9:37-43
Expand Messages
- passage: Gal 1:3-1:10
Chapter 1
Grace be to you and peace from God the Father, and from our Lord Jesus
Christ, Who gave himself for our sins, that he might deliver us from this
present evil world, according to the will of God and our Father: To whom
be glory for ever and ever. Amen. I marvel that ye are so soon removed
from him that called you into the grace of Christ unto another gospel:
Which is not another; but there be some that trouble you, and would
pervert the gospel of Christ. But though we, or an angel from heaven,
preach any other gospel unto you than that which we have preached unto
you, let him be accursed. As we said before, so say I now again, if any
man preach any other gospel unto you than that ye have received, let him
be accursed. For do I now persuade men, or God? or do I seek to please
men? for if I yet pleased men, I should not be the servant of Christ.
passage: Luke 9:37-9:43
Chapter 9
And it came to pass, that on the next day, when they were come down
from the hill, much people met him. And, behold, a man of the company
cried out, saying, Master, I beseech thee, look upon my son: for he is
mine only child. And, lo, a spirit taketh him, and he suddenly crieth
out; and it teareth him that he foameth again, and bruising him hardly
departeth from him. And I besought thy disciples to cast him out; and
they could not. And Jesus answering said, O faithless and perverse
generation, how long shall I be with you, and suffer you? Bring thy
son hither. And as he was yet a coming, the devil threw him down, and
tare him. And Jesus rebuked the unclean spirit, and healed the child,
and delivered him again to his father. And they were all amazed at the
mighty power of God. But while they wondered every one at all things
which Jesus did, he said unto his disciples,
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text:
Welcome to KYOTO SAGANO WALK & BAMBOO FOREST
Walking Tour (Half Day)
Every Thursday from March to November (no tours in Dec, Jan & Feb)
10:00-13:00 (about 3 hours, no lunch)
Price : 2,000 yen/person
1,000 yen/child (age 15 and under)/free of charge (up to 2 children accompanied by 1 adult)
* 300 yen for entrance fee at Otagi-nenbutuji temple and 230 yen for bus fare are also needed (You can use Kyoto city bus All- day Pass)
No need to make a reservation. You can join the tour just by coming to the meeting place.
But if you wish to make a reservation, you can send email to email@example.com
JR Saga-Arashiyama Station (10:00)
Take Kyoto bus (about 7 min.)
Otagi-nenbutsuji temple (entrance fee 300 yen)
Preserved town area of Saga-Toriimoto
Bamboo grove
Nonomiya shrine (we dismiss here)
* This tour is not covered by insurance, so please participate in this tour at your own risk.
Meeting point
JR Saga-Arashiyama Station outside the ticket gate
* You can use JR Pass to get to JR Saga-Arashiyama Station.
Private Tours
Private tours are also available for 15,000 yen (4 hours) and 30,000 yen (8 hours)
For inquiry, please send email to email@example.com
Please send us your comment to our Facebook page


text:
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S-adenosyl-L-methionine (SAM) is synthesized by methionine adenosyltransferases (MATs). Ablation of the liver-specific MAT1A gene results in liver neoplasia and sensitivity to oxidant injury. Here we show that acidic sphingomyelinase (ASMase) mediates the downregulation of MAT1A by TNF-α. The levels of MAT1A mRNA as well as MAT I/III protein decreased in cultured rat hepatocytes by in situ generation of ceramide from exogenous human placenta ASMase. Hepatocytes lacking the ASMase gene (ASMase–/–) were insensitive to TNF-α but were responsive to exogenous ASMase-induced downregulation of MAT1A. In an in vivo model of lethal hepatitis by TNF-α, depletion of SAM preceded activation of caspases 8 and 3, massive liver damage, and death of the mice. In contrast, minimal hepatic SAM depletion, caspase activation, and liver damage were seen in ASMase–/– mice. Moreover, therapeutic treatment with SAM abrogated caspase activation and liver injury, thus rescuing ASMase+/+ mice from TNF-α–induced lethality. Thus, we have demonstrated a new role for ASMase in TNF-α–induced liver failure through downregulation of MAT1A, and maintenance of SAM may be useful in the treatment of acute and chronic liver diseases.
TNF-α plays a central role in many forms of liver injury (1–3) and hence information on signaling of hepatocellular cell death by TNF-α may be of therapeutic relevance. The signaling mechanism mediating the intracellular effects of TNF-α is complex and can lead to multiple responses, including proliferation, inflammation, or cell death. The cytotoxic effects of TNF-α are the result of the generation of death signals that originate from the binding of TNF-α to the TNF-α–R1 receptor, a typical death receptor with a 60– to 80–amino acid cytoplasmic sequence known as the death domain. Research in this area has unveiled complex protein-protein interactions, recruitment of signaling intermediates, and activation of proteins that mediate the apoptotic effect of TNF-α (4, 5). Ceramide has emerged as a putative signaling lipid mediator that plays a role in the stress response, cell death, and TNF-α signaling (6–9). Ceramide levels can increase by de novo synthesis through activation of serine-palmitoyl transferase, the rate-limiting enzyme in ceramide synthesis, or ceramide synthetase (10, 11). However, the hydrolysis of sphingomyelin upon activation of sphingomyelinases (SMases) constitutes an effective means for the rapid generation of ceramide (8, 9). The binding of TNF-α to its plasma membrane receptor TNF-α–R1 results in the activation of two major types of SMases: a neutral SMase (NSMase), with an optimum pH around 7.5, which is membrane bound and Mg2+ dependent; and an acidic SMase (ASMase), with an optimum pH of about 4.8, which is further subclassified into two isoforms, an endosomal/lysosomal ASMase and a secretory Zn2+-dependent ASMase (8, 9, 12, 13).
While activation of these enzymes may generate ceramide at different cellular sites that mediate the diverse effects of TNF-α, their role in apoptosis is not well established. Thus, NSMase has been reported to be involved in the stress response mediating the cytotoxic effects exerted by chemotherapeutic agents (14), whereas ASMase has been shown to play an essential role in ionizing radiation–mediated cell death (15, 16) as well as in the developmental death of oocytes (17). In addition, the factor associated with NSMase activation, an adaptor protein involved in NSMase activation, has been shown to contribute to TNF-α–induced fibroblast apoptosis (18), although the role of NSMase ablation itself in TNF-α–induced apoptosis remains unknown. On the other hand, the role of ASMase in Fas-induced cell death appears to be restricted to hepatocytes, as the absence of ASMase in hepatocytes has been shown to confer resistance to Fas-mediated cell death (19, 20), while other cell types from ASMase knockout mice are sensitive to Fas (19). Moreover, we have recently demonstrated that ASMase plays a significant role in hepatocellular apoptosis and liver damage induced by TNF-α (21).
The liver plays a prominent role in the synthesis and consumption of S-adenosyl-L-methionine (SAM, also abbreviated as "AdoMet"), which functions as the one-carbon donor for methylation of DNA, RNA, and proteins and in the synthesis of biogenic amines and glutathione (GSH) (22–24). Furthermore, SAM is also required for DNA and RNA methylation in mitochondria and as an intermediate in the biosynthesis of ubiquinone and for sterol methylation (25, 26). SAM is synthesized from methionine and ATP by methionine adenosyltransferases (MATs). In mammals there are three known MAT isoforms (MAT I, MAT II, and MAT III) (23, 27, 28); MAT I and MAT III are a tetramer and a dimmer, respectively, of the same α1 subunit, a 43.7-kDa polypeptide encoded by the gene MAT1A, which is exclusively expressed in adult liver (23). MAT2A, on the other hand, is ubiquitously expressed and encodes a catalytic subunit (α2) found in a native MAT isoenzyme (MAT II) in association with a regulatory β subunit (29). In the liver, impairment of MAT I/III results in lowered SAM levels, which contributes to the progression of liver injury in experimental models and in patients with alcohol- or virus-induced cirrhosis (23, 30–33). Indeed, the most compelling evidence for the relevance of hepatic SAM has been recently documented with the development of MAT1A knockout mice (34). This paradigm illustrates the devastating consequences of chronic hepatocellular SAM depletion, which enhances the susceptibility of the liver to oxidant-induced cell death, metabolic disturbances, and spontaneous hepatocellular carcinoma (34, 35).
A common strategy for investigating the cellular effects of ceramide has been the incubation of cells with exogenous SMases, cell-permeable analogs, or natural ceramide, which has revealed a wide spectrum of responses ranging from cell cycle arrest to proliferation, oxidative stress, apoptosis, and gene regulation (6, 8, 21, 36, 37). As for the effect on gene regulation, membrane-permeant ceramide analogs (C2 and C6) as well as exogenous NSMase have been shown to modulate the expression of specific genes such as cytochrome P450 2C11, α1-acid glycoprotein, or γ-glutamylcysteine synthetase (γ-GCS) in rat hepatocytes, mimicking the effects of interleukin-1 or TNF-α (36, 37). In addition, we have recently reported that the cell-permeable ceramide C2 downregulated MAT1A expression in H35 rat hepatoma cells (38). Thus, the present study was undertaken to examine the regulation of MAT1A by individual SMases, namely Bacillus cereus NSMase (bNSMase) and human placenta ASMase (hASMase), previously characterized in rat hepatocytes (37), with the ultimate goal of assessing the contribution of ASMase to the regulation of MAT1A expression and to test the therapeutic role of SAM in TNF-α–induced fulminant liver failure.
ASMase knockout mice (C57BL/6 strain) were propagated using heterozygous breeding pairs (a generous gift from R. Kolesnick, Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, USA; and E. Gulbins, University of Duisburg-Essen, Essen, Germany) and were genotyped as described previously (21). Hepatocytes from male Sprague-Dawley rats (200–300 grams) or male mice (8–12 weeks old) were isolated by collagenase perfusion with flow rates of 16–18 ml/min and 7–9 ml/min, respectively, and were cultured at a density of 2 × 106 cells on 60-mm dishes coated with rat tail collagen. Rat or mouse hepatocytes were incubated with recombinant human TNF-α (15–280 ng/ml; Preprotech EC, London, United Kingdom) or exogenous SMases (Sigma-Aldrich, St. Louis, Missouri, USA) by dilution of concentrated hASMase (250 U/0.55 ml in 50% glycerol, 25 mM potassium phosphate, pH 4.5, 0.1% Triton X-100, and 0.05 mM PMSF) and bNSMase (100 U/ml in 50% glycerol and 50 mM Tris-HCl, pH 7.5) with culture medium to a concentration of 0.1–1.0 U/ml as described previously (37). Some cultures were pretreated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol HCl (D-threo-PDMP), N-butyldeoxynojirimycin, bafilomycin A1, chloroquine, or mannose 6-phosphate (M 6-P) before the addition of TNF-α or hASMase. Alternatively, cells were incubated with cell-permeable ceramide (C2 or C6), ganglioside GD3 (GD3), ganglioside GM3 (GM3), glucosylceramide, lactosylceramide, or lyso-glucosylceramide (1–5 μM) (Matreya, Pleasant Gap, Pennsylvania, USA) in ethanol (0.1%). In addition, cells were incubated with the natural ceramide C16 in dodecane/ethanol (2:98 [volume/volume]; 0.05% final concentration) or solvent only as a control. The experimental protocols met the guidelines of and were approved by the Animal Care Committee of the Hospital Clinic-Universidad de Barcelona.
MAT1A and MAT2A mRNA levels were determined by real-time RT-PCR analyses using a LightCycler and a Faststart SYBR Green I DNA quantification kit (Roche, Mannheim, Germany) following the manufacturer's protocol. cDNA was prepared from total RNA (2 μg) at 50°C for 50 minutes using random hexamers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, USA) following the manufacturer's protocol. The primers used were: MAT1A, forward, 5′-ATGGACCTGTGGATGGC-3′, and reverse, 5′-TGGCCTCCAGTGTTATGT-3′; MAT2A, forward, 5′CCATGAGGCGTTCATCG-3′, and reverse, 5′-GCGTAACCAAGGCAATG-3′; 18S, forward, 5′-GGGGAATCAGGGTTCGATT-3′, and reverse, 5′-GCGCCGGTCCAAGAATTTCA-3′. The second-derivative maximum method was taken to determine the crossing points automatically for individual samples, and relative amounts of target genes were calculated based on the crossing-point analysis. The result was expressed as a ratio of product copies to copies of housekeeping gene 18S from the same RNA.
MAT1A and MAT2A mRNA levels were also determined by Northern blot analyses of total RNA isolated using the Trizol reagent (Gibco/BRL, Grand Island, New York, USA). Ten micrograms of RNA were separated by electrophoresis under denaturing conditions and were transferred onto Hybond-XL nylon membranes (Amersham Biosciences, Uppsala, Sweden) and hybridized to random-primed 32P-labeled cDNA probes.
Samples were resolved by 12.5% SDS-PAGE, transferred to nitrocellulose membrane, and incubated with rabbit anti–MAT I/III (1:10,000 dilution), a generous gift from M. Pajares (Instituto Investigaciones Biomédicas Alberto Sols, Consejo Superior Investigaciones Científicas, Madrid, Spain), characterized previously (39). Membranes were incubated with peroxidase-conjugated secondary antibodies (1:5,000 dilution; Amersham, Arlington Heights, Illinois, USA), and bound antibody was visualized with ECL detection (ECL; Amersham) on Kodak X-OMAT film (Eastman Kodak, Rochester, New York, USA).
SAM or SAH levels were determined in liver homogenates (or in mitochondrial fractions isolated from liver homogenates) after homogenization with 0.5 M HClO4 followed by neutralization of the sample with 3 M KOH. SAM/SAH levels were determined by HPLC using a Partisil SCx 10-μm column by isocratic elution at a flow rate of 1 ml/min with 0.03 mol/l NH4H2PO4 containing 2% acetonitrile, pH 2.6, as described before (33). SAM and SAH peaks were identified at 254 nm by sample spiking with authentic standards. The amount of either metabolite was calculated from standard curves relating the peak area to calibration curves with standards. In some cases, mitochondria were isolated by Percoll gradient centrifugation as described (40). GSH levels in homogenates or mitochondria were determined using HPLC (21, 37).
The combination of D-(+)-galactosamine (Gal) plus TNF-α (Gal/TNF) or Gal plus LPS (Gal/LPS) is a known model of lethal hepatitis (41, 42). C57Bl/6 mice were used at an age of 8–10 weeks. A single dose of recombinant murine TNF-α (10 mg/kg intravenously; Preprotech EC, London, United Kingdom) or LPS (50 μg/kg intraperitoneally; Escherichia coli serotype 0128:B12; Sigma-Aldrich) was administered after Gal treatment (700 mg/kg intraperitoneally), resulting in the death of mice (90–95%) within 6–8 hours. In some cases, SAM (p-toluensulfonate form; Europharma, Madrid, Spain) was given hourly for 8 hours after TNF-α or LPS injection (5 mg/mouse intraperitoneally). For estimation of the effect of prolonged SAM treatment, SAM was administered to Gal/TNF- or Gal/LPS-treated mice for 24 hours (hourly for the first 8 hours and then every 8 hours). For blocking of GSH synthesis, L-buthionine sulfoximine (BSO) was administered (4 mmol/kg) 30 minutes before and 3 hours after TNF-α or LPS administration. Alternatively, N-acetylcysteine (NAC) (30 mg/mouse intraperitoneally) was given to mice before treatment with Gal/TNF or Gal/LPS to assess the efficacy of antioxidants on the course of liver injury. For histology, liver specimens were washed with PBS and embedded in paraffin. Sections 7 μm in thickness were stained with hematoxylin and eosin using standard methods. Serum alanine aminotransferase (ALT) and aspartate aminotransferase levels were determined by a colorimetric test (Sigma-Aldrich).
Paraffin-embedded liver tissue was cut into slices 4 μm in thickness using a microtome (Leica RM2155; Leica Instruments, Bensheim, Germany) and was fixed to glass slides. The TUNEL assay was performed by incubation of the slices with 2 μg/ml proteinase K (Sigma-Aldrich) in 10 mM Tris-HCl buffer containing 5 mM EDTA at a pH of 7.4. Sections were then labeled following the instructions of a commercial Kit (In situ Cell Death Detection Kit; Roche Diagnostics), stained with 3,3′-diaminobenzidine (Roche Diagnostics), and counterstained with hematoxylin and eosin. The slides were mounted and examined using a Nikon Eclipse E1000 microscope.
Liver was homogenized in a buffer containing 120 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0,5 % Igepal, 2 mM ethylene glycol-bis-(β-aminoethyl ether) tetraacetic acid, and 50 μM PMSF, incubated at 4°C for 15 minutes with shaking, and spun down at 12,000 g at 4°C for 15 minutes. Caspase activity was assayed with 200 μg of cell lysate and 1.25 ml of assay buffer containing 100 mM NaCl, 10% sucrose, 0.1% (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 10 mM DTT, pH 7.4, by the release of 7-aminomethyl coumarin from 40 nmol of Ac-DEVD-AMC (caspase 3) or Ac-IETD-AMC (caspase 8) (Calbiochem, San Diego, California, USA). Fluorescence was continuously recorded with emission at 460 nm and excitation at 380 nm as described (43).
In addition, caspase activation was also monitored by immunoblotting using rabbit anti–caspase 3 (1:1,000 dilution; Pharmingen, San Diego, California, USA) and anti–caspase 8 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, California, USA) as primary antibodies. Membranes were then incubated with peroxidase-conjugated secondary antibodies (Amersham) and bound antibodies were visualized as described above for MAT I/III.
Gangliosides levels (including GM1, GM3, and GD3) were measured by high-performance thin-layer chromatography as described previously (43). Ceramide levels were quantified by HPLC (37).
Total TNF-α (bound and free) was measured in serum after Gal/LPS treatment using a mouse-specific competitive enzyme immunoassay kit (Chemicon, Temecula, California, USA).
Statistical analyses of mean values ± SD for multiple comparison were made by one-way analyses of variance followed by Fisher's exact test. Survival curves were compared using a log-rank χ2 test.
Preservation of high levels of MAT1A is critical for a differentiated and functional competent liver. Upon isolation from the intact organ, cultured rat hepatocytes undergo a gradual decline in MAT1A mRNA as it is replaced by MAT2A mRNA (23, 44). In vitro stabilization of MAT1A, however, can be achieved by the incubation of cultured hepatocytes with SAM (44). Because TNF-α downregulates MAT1A expression in cultured rat hepatocytes (38) and SMases mediate TNF-α signaling (8, 9), we first examined the role of individual SMases on MAT1A expression. Cultured rat hepatocytes were incubated with exogenous bNSMase and hASMase, and the levels of MAT1A and MAT2A mRNA were examined over time. Although the intracellular levels of ceramide generated by exposure of cultured hepatocytes to exogenous bNSMase or hASMase were equivalent (2–4 nmol ceramide/mg cellular protein), these enzymes regulated the expression of MAT1A and MAT2A in a divergent fashion. In the presence of exogenous SAM (3 mM), the level of MAT1A mRNA in control hepatocytes remained unchanged for 14–16 hours (not shown) in agreement with previous results (44). Next, we examined the effect of exogenous SMases on MAT1A mRNA by Northern blot and real-time PCR analyses. Either approach showed equivalent results, indicating a decrease in the levels of MAT1A mRNA by hASMase while those of MAT2A mRNA increased, thus mimicking the effect observed with TNF-α (Figure (Figure1,1, A and B). In contrast, exogenous bNSMase did not affect the expression of MAT1A or MAT2A (Figure (Figure1,1, A and B). The repressive effect of hASMase was observed within 3 hours of incubation, with a significant reduction in MAT1A mRNA levels to 39–45% of control values. To determine if the downregulation of MAT1A mRNA was reflected at the protein level, we analyzed the MAT I/III protein content by Western blot. As seen, both TNF-α and hASMase but not bNSMase reduced the levels of MAT I/III (Figure (Figure1C).1C). Consistent with these findings, we confirmed a severe reduction in MAT I/III enzymatic activity (8.7 ± 1.2 vs. 3.2 ± 0.8 pmol/min/mg protein in control or hASMase-treated cells, respectively; P < 0.05). Moreover, as TNF-α increases the cellular levels of glycosphingolipids synthesized in the Golgi from ceramide, and as glycosphingolipids play an important role in cell death (21, 43), we next examined the contribution of glycosphingolipids in the regulation of MAT1A by TNF-α. In agreement with previous results (21), preincubation of cells with D-threo-PDMP inhibited glucosylceramide synthase (GCS), the first enzyme in glycosphingolipid synthesis, decreasing the stimulation of GD3 and GM1 levels by TNF-α (325% ± 56% or 298% ± 76% vs. 8% ± 5% or 3% ± 4% for GD3 and GM1 in the absence or presence of D-threo-PDMP, respectively; P < 0.05). As expected, D-threo-PDMP enhanced TNF-α–generated ceramide levels (2.3 ± 0.3–fold; P < 0.05). However, under these conditions the downregulating effect of TNF-α on MAT1A expression was not affected by D-threo-PDMP (Figure (Figure1A).1A). Similar findings were observed using N-butyldeoxynojirimycin, another GCS inhibitor (not shown). Thus, these findings show the ability of ceramide generated specifically from hASMase to downregulate the expression of MAT1A and exclude the possibility of a role for glycosphingolipids in this effect.
We have previously shown that exogenous hASMase enters hepatocytes by endocytosis being delivered to intracellular acidic compartments (37). To assess whether hASMase requires acidic vesicles for its effect on MAT1A expression, we next determined the effect of lysosomotropic agents that abolish acidification of intracellular vesicles. As shown (Figure (Figure2),2), bafilomycin A1, an inhibitor of the H+-ATPase pump responsible for acidifying lysosomes and late endosomes (45), and chloroquine, known to disrupt acidic compartments (46), each prevented the downregulation of MAT1A induced by hASMase (Figure (Figure2).2). Moreover, M 6-P, which blocks the delivery to endosomes/lysosomes of enzymes via the M 6-P receptor (47), abolished the reduction in MAT1A mRNA by hASMase (Figure (Figure2).2). A similar effect for bafilomycin A1 and chloroquine was observed in the TNF-α–mediated downregulation of MAT1A mRNA levels (not shown). Thus, these findings indicate that the internalization of hASMase into acidic vesicles is required for the downregulation of MAT1A mRNA.
To assess the structural requirement of ceramide, we tested the role of sphingolipid analogs on MAT1A expression. As seen, membrane-permeant ceramide C2, as well as glycosphingolipids GD3, GM3, lactosylceramide, and glucosylceramide, reduced MAT1A mRNA levels to a similar extent while increasing those of MAT2A (Figure (Figure3).3). Similar findings were observed for MAT1A regulation using the natural ceramide C16 (not shown). In contrast, incubation of cells with sphingomyelin, sphingosine, or dihydroceramide C2 did not change the relative levels of MAT1A or of MAT2A mRNA. In addition, incubation of cells with lyso-lactosylceramide (Matreya) did not affect the level of MAT1A or MAT2A mRNA (not shown). Thus, taking into account that carbohydrates are present only in glycosphingolipids but not in sphingolipids and that the N-fatty acyl-sphingosine moiety and the trans-4,5 double bond are structural features common to both lipid classes, it appears that the carbohydrate groups are not required for the regulation of MAT1A expression.
To assess the specific role of ASMase in the regulation of MAT1A by TNF-α, we used hepatocytes from ASMase–/– mice, as early signaling events in response to TNF-α are preserved in ASMase knockout mice (21). Compared with those in ASMase+/+ cells, the levels of MAT1A mRNA in hepatocytes lacking the ASMase gene were unaffected upon exposure to TNF-α (Figure (Figure4,4, A and B). However, despite the unresponsiveness of ASMase–/– hepatocytes to TNF-α exposure, the addition of exogenous hASMase markedly decreased the levels of MAT1A mRNA while increasing those of MAT2A (Figure (Figure4B).4B). This effect was specific for ceramide derived from hASMase, as incubation of cells with exogenous bNSMase did not affect MAT1A or MAT2A mRNA content (Figure (Figure4).4). These results show that the repressive effect of TNF-α on MAT1A expression is mediated through the generation of ceramide from ASMase.
To address whether the contribution of ASMase to TNF-α–induced liver injury is through MAT1A downregulation and subsequent SAM decrease, we examined the changes over time of hepatic SAM produced by Gal and TNF-α or LPS in a well-established model of lethal hepatitis (41, 42). In ASMase+/+ mice (n = 25), Gal/TNF caused a time-dependent decrease of the hepatocellular SAM levels before the occurrence of liver damage, as monitored by the rise in ALT in serum (Figure (Figure5A).5A). Histological examination of liver samples from Gal/TNF- or Gal/LPS-treated mice indicated extensive hemorrhagic lesions with clusters of damaged cells (Figure (Figure5B).5B). TUNEL assay showed the presence of apoptotic hepatocytes in liver specimens from Gal/LPS-treated wild-type mice (Figure (Figure6A).6A). In contrast, the hepatic levels of SAM in ASMase–/– mice treated with Gal/TNF remained significantly higher than those in wild-type mice, and this outcome was accompanied by minimal liver damage, as monitored by serum transaminases (Figure (Figure5A),5A), liver histology (Figure (Figure5B),5B), and TUNEL assay (Figure (Figure6A).6A). Northern blot analyses indicated a decrease in MAT1A mRNA levels in ASMase+/+ mice by 3 hours after Gal/TNF treatment (not shown). The hepatocellular levels of SAM in the ASMase knockout mice after Gal/TNF treatment were similar to those in ASMase+/+ mice treated with Gal alone (63 ± 5 nmol/g liver and 53 ± 6 nmol/g liver 3 hours and 6 hours after, respectively). As this model of liver injury is caspase dependent, we examined the activation of caspases 8 and 3 in liver samples after Gal/TNF administration by a fluorescence assay using appropriate substrates and by immunoblot. Although each approach showed activation of caspases 3 and 8 by Gal/TNF in hepatocytes from wild-type mice (Figures (Figures5C5C and and6,6, B and C), interestingly, caspase activation was much lower in ASMase–/– mice treated with Gal/TNF than in ASMase+/+ mice (Figures (Figures5C5C and and6,6, B and C), suggesting that ASMase is necessary for optimal caspase activation in this model. Furthermore, as hepatic SAM regulates GSH homeostasis, which in turn can modulate caspase activation (48, 49), we next determined the time course of hepatic GSH levels in this model of hepatocellular cell death. As seen, hepatic GSH levels decreased in ASMase+/+ mice over time, lagging behind the reduction in SAM content in ASMase+/+ mice but not in ASMase–/– mice (Figure (Figure5,5, A and D). Similar results were observed with Gal/LPS (not shown). Finally, the levels of mitochondrial GSH, an essential pool of GSH in the control of mitochondrial function and cell death (21, 40), were reduced to 51 ± 7% of control levels in ASMase+/+ mice (Figure (Figure55E).
Because the fall in SAM levels was a primary event in this model of liver failure, we next examined the therapeutic role of SAM in ASMase+/+ mice. Initial experiments indicated that a single intraperitoneal injection of 10 mg/kg of SAM, a dose previously used in experimental models of liver injury and fibrogenesis (33, 50), before Gal/TNF or Gal/LPS administration was ineffective in preserving hepatic SAM levels and in preventing liver damage (not shown). However, continuous SAM treatment, hourly after Gal/LPS exposure, prevented liver injury through restoration of hepatic SAM (Figure (Figure7,7, A–C). Furthermore, SAM treatment decreased caspase activation, as assessed by immunoblot (Figure (Figure6,6, B and C) or stimulated fluorescence (Figure (Figure7D),7D), and these effects were accompanied by restoration of intracellular GSH stores in cytosol (Figure (Figure7E)7E) and mitochondria (Figure (Figure5E).5E). Indeed, the specific contribution of GSH to the protective effect of SAM was further assessed by treatment of mice with BSO, an inhibitor of γ-GCS. Pretreatment of ASMase+/+ mice with BSO after Gal/LPS administration abolished the protective effect of SAM treatment (Figure (Figure7,7, A and B). BSO treatment, however, did not affect the levels of SAM but prevented the recovery of GSH (not shown). In addition, to ensure that the protective effect of SAM was not due to reduced TNF-α secretion caused by LPS, we monitored plasma TNF-α levels over time. As seen, the time course of TNF-α appearance in plasma was similar in Gal/LPS-treated mice with or without SAM therapy (Figure (Figure7F).7F). Moreover, we checked the safety of chronic SAM therapy in Gal/LPS-treated mice. As seen, sustained administration of SAM was safe and devoid of side effects, as mice were protected from Gal/LPS-induced liver damage (Figure (Figure8A).8A). In addition, NAC, an antioxidant, reduced the liver injury caused by Gal/LPS (Figure (Figure88B).
Finally, the protective role of SAM "translated" to an improved survival rate (Figure (Figure9).9). As seen, mice (n = 15) treated with SAM were significantly protected from lethality, compared with the control group treated with saline. Surviving mice were still alive for at least 5 days after the Gal/LPS treatment. Treatment of mice with SAM for 24 hours resulted in a similar survival rate (not shown). Similar results in terms of survival were observed in mice treated with Gal/TNF instead of LPS. In addition, 80–90% of ASMase–/– mice survived at least 5 days after Gal/TNF treatment.
The present study reveals a novel mechanism of action of ASMase whereby this signaling enzyme promotes TNF-α–induced hepatocellular damage by decreasing the expression of MAT1A. Our first approach was the use of exogenous hASMase. Certain structural features of this enzyme have been characterized in comparison with those of recombinant human ASMase (51). Those studies demonstrated that recombinant human ASMase and the native protein purified from human placenta showed a similar glycosylation pattern and the presence of a critical disulfide bond in the sphingolipid activator N-terminal domain. In addition, prior studies demonstrated that hASMase enters cells including hepatocytes via M 6-P receptor–mediated endocytosis and is delivered into acidic compartments (37, 47, 52). Consistent with the pH optima of hASMase, we verified that agents that block intracellular vesicle acidification abolished the effect of hASMase on MAT1A expression. In addition, the action of hASMase on MAT1A expression was prevented by an excess of M 6-P, which would be expected to compete with hASMase for endocytosis without dissipating the pH gradient of acidic intracellular compartments. Thus, based on these findings it appears that endocytosis of hASMase and its delivery into acidic vesicles where the enzyme becomes active are required for the downregulation of MAT1A expression. However, the precise intracellular site where hASMase becomes activated remains to be identified. In addition to this approach, we also used hepatocytes deficient in ASMase–/–. The data we obtained show the resistance of hepatocytes from ASMase knockout mice to TNF-α–induced downregulation of MAT1A. However, the levels of MAT1A mRNA were reduced by hASMase, indicating that the response of hepatocytes from ASMase knockout mice to TNF-α in the regulation of MAT1A is dependent on the presence of ASMase.
An important implication from these results is the divergent functions of NSMase versus ASMase in the regulation of MAT1A expression. Not only did bNSMase, a Mg2+-dependent exogenous SMase at neutral pH, characterized previously (8, 9, 37), fail to affect the expression of MAT1A, but also, more importantly, the activation of NSMase by TNF-α remains intact in hepatocytes from ASMase knockout mice (21) and yet in ASMase–/– hepatocytes, TNF-α failed to downregulate MAT1A. Thus, our findings clearly indicate that the ceramide pool signaling the downregulation of MAT1A originates from an acidic intracellular compartment, whereas the ceramide pool derived from NSMase is inactive, illustrating that the role of ceramide in cell signaling depends on the specific SMase activated. This outcome regarding the effect of ASMase on gene regulation contrasts with previous reports discarding a role for ceramide produced in lysosomes in apoptosis signaling. For instance, stress-induced apoptosis was observed in Epstein-Barr virus–transformed B lymphocytes from control individuals as well as from patients affected with Niemann-Pick disease, an autosomal recessive condition characterized by deficient ASMase in lysosomes (14). In contrast, DeMaria et al. (52), who also used Niemann-Pick lymphoblast lines, concluded that ASMase was required for Fas-mediated apoptosis. On the other hand, hepatocytes deficient in ASMase have been shown to be resistant to ionizing radiation–, Fas- or TNF-α–mediated cell death (15, 19–21). Furthermore, consistent with our findings regarding MAT1A regulation, Monney et al. (53) reported that agents that alkalinize acidic vesicles attenuate TNF-α–induced apoptosis in U937 human monocytes and L292 mouse fibroblasts. Thus, it appears that the role of individual SMases in signaling pathways (apoptosis or gene regulation) depends on several conditions, such as the stimuli used and type of cell studied. Hence, taken collectively, our data establish that MAT1A is selectively downregulated by ceramide generated from the activation of ASMase in acidic intracellular compartments, which mediates the effects observed with TNF-α.
Of note, we would like to emphasize that the downregulating effect of TNF-α or hASMase on MAT1A mRNA levels was observed despite the presence of exogenous SAM, which stimulates the transcription of MAT1A (44). Thus, in further examining the putative mechanism involved in the downregulation of MAT1A by ASMase, we determined the stability of MAT1A mRNA in the presence of actinomycin D. Our data show that the presence of hASMase shortened the half-life of MATA1 mRNA from 5.4 ± 0.3 hours to 3.3 ± 0.2 hours (P = 0.005). In an attempt to characterize this process further, we observed that cycloheximide pretreatment abolished the effect of hASMase on MAT1A mRNA decay, suggesting the involvement of specific proteins. Thus, although at present we do not understand the mechanism involved in the posttranscriptional decay of MAT1A mRNA by ASMase, an important pathway in the control of mRNA includes the recruitment of the exosome by AU-binding proteins to affect 3′-to-5′ mRNA degradation (54). Although clearly more work will be required to fully characterize this novel mechanism in the regulation of MAT1A expression by ASMase-induced ceramide generation, including the identification of the specific proteins involved and the role of the AU site in the 3′ untranslated region of MAT1A, similar effects on the modulation of mRNA stability have been described recently for cell-permeable ceramides in other cell types. For instance, in Jurkat cells, ceramide C2 shortened the half-life of Bcl-2 mRNA through a conserved AU-rich element in the 3′ untranslated region of Bcl-2 mRNA (55). In addition, other remaining questions include how ceramide recruits or activates this regulated pathway (54). Whether ceramide generated within acidic vesicles undergoes a transbilayer movement similar to what has been recently reported for bNSMase (56) in erythrocyte ghosts remains to be established. In hepatocytes we have shown that the ceramide pool generated from bNSMase must undergo a "flip-flop" movement across the bilayer to signal the transcriptional regulation of genes such as γ-GCS (37).
To examine the in vivo consequences of the downregulation of MAT1A by ASMase, we analyzed the regulation of hepatic SAM during the progression of liver failure in a model of lethal hepatitis. This approach revealed that the depletion of hepatocellular SAM levels precedes the activation of caspases induced by TNF-α, resulting in liver damage and lethality. The outcome of ASMase–/– mice challenged with Gal/TNF was similar to that of ASMase+/+ mice treated with Gal alone. In both cases, the mild decrease in hepatic SAM reflects the effect of Gal, a hepatotoxin that blocks transcription in hepatocytes. MAT I/III is sensitive to oxidative stress due to the targeting of a critical cysteine residue (30, 32). Hence the downregulation of MAT1A and the inactivation of MAT I/III accelerate the depletion of SAM by TNF-α in ASMase+/+ mice, initiating a cascade of events that culminate in the death of mice. As shown by TUNEL assay, the fulminant outcome of this model reflects a prominent hepatocellular apoptosis. In comparing caspase activation in ASMase–/– versus ASMase+/+ mice, we unexpectedly observed a defective activation of caspase 8 by TNF-α in ASMase knockout mice. Previous findings reported the involvement of upstream caspases, such as caspase 8, in the activation of ASMase (57–59); however, our data indicate that ASMase is needed for optimal caspase 8 activation by TNF-α (Figure (Figure5C)5C) and suggest an inverse hierarchical ordering in caspase 8/ASMase activation, as reported for Fas (58, 59). This effect seems to be dependent on SAM through replenishment of GSH levels, as SAM therapy attenuated caspase 8 activation in ASMase+/+ mice (Figure (Figure7D).7D). Furthermore, our findings also indicate the regulation of caspase 3 by ASMase. In the absence of ASMase, we observed a lowered activation of caspase 3 by TNF-α (Figures (Figures5C5C and and6B),6B), and this ASMase-independent caspase 3 activation may be due to the participation of other signaling proteins, such as cathepsin B (5). In addition, SAM seems to regulate caspase 3 activation. This outcome may reflect the ability of SAM to regulate the mitochondrial commitment to cell death (60, 61). Depletion of SAM in the livers of MAT1A–/– knockout mice has been shown to modulate mitochondrial integrity through regulation of the expression of prohibitin, a protein encoded in the nucleus that participates in the functional organization of mitochondria (60). However, in addition to this potential mechanism, it is likely that the protective effect of SAM in lethal hepatitis is dependent on the replenishment of GSH levels, particularly in mitochondria (39, 62), as BSO pretreatment abrogated the therapeutic role of SAM. Indeed, mitochondrial GSH has been shown to play an essential role in the survival of hepatocytes in the face of TNF-α–mediated cell death (21, 37, 40, 62).
The present study complements recent observations on the mechanisms of TNF-α–induced hepatocellular damage (21) (Figure (Figure10).10). Through MAT1A downregulation and subsequent SAM decrease, mitochondrial GSH levels become limited, leaving hepatocytes unprotected from death signals generated by TNF-α–induced ASMase activation, such as GD3 (21). As GD3 can target mitochondria (63), the consequences of this interaction, which leads to apoptosome assembly and caspase 3 activation, depend largely on the levels of mitochondrial GSH (21, 43, 63). Thus, the combination of mitochondrial GSH depletion and the trafficking of GD3 to mitochondria mediates the hepatocellular damage of TNF-α. An implication of the current and previous findings (21) is that in addition to the therapeutic effect of SAM, interference with ASMase signaling may be beneficial, as it would spare SAM/GSH levels and avoid the production of harmful lipids such as GD3. The effects of ASMase inhibitors in vivo on liver function and protection against liver failure remain to be established, however.
Taken together, our findings characterize a novel function of ASMase in the cytotoxicity of TNF-α. Given that TNF-α is essential in several forms of liver damage, such as alcohol-induced liver injury (31, 40) and lethal hepatitis (41, 42), our findings may be of therapeutic relevance for the prevention of fulminant liver failure and the treatment of liver diseases through replenishment of SAM and subsequent restoration of mitochondrial GSH.
We want to express our gratitude to R. Kolesnick and E. Gulbins for the gift of ASMase knockout mice, and M. Pajares for anti–MAT I/III. The work presented was supported in part by the Research Center for Liver and Pancreatic Diseases, P50 AA 11999; and grant 1R21 AA014135-01, funded by the US National Institute on Alcohol Abuse and Alcoholism; Plan Nacional de I+D grants SAF01-2118, SAF2002-3564, and SAF2003-04974; and Red Temática de Investigación Cooperativa G03/015 and Red de Centros C03/02, supported by Instituto de Salud Carlos III. We want to thank Susana Nuñez for expert technical assistance and genotyping of ASMase–/– mice. M. Marí and A. Morales are Ramón y Cajal investigators.
Nonstandard abbreviations used: acidic sphingomyelinase (ASMase); alanine aminotransferase (ALT); Bacillus cereus sphingomyelinase (bNSMase); d-(+)-galactosamine (Gal); d-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol HCl (d-threo-PDMP); ganglioside GD3 (GD3); ganglioside GM3 (GM3); glucosylceramide synthase (GCS); γ-glutamylcysteine synthetase (γ-GCS); glutathione (GSH); human placenta sphingomyelinase (hASMase); l-buthionine sulfoximine (BSO); liver-specific methionine adenosyltransferase (MAT1A); mannose 6-phosphate (M 6-P); methionine adenosyltransferase (MAT); N-acetylcysteine (NAC); neutral sphingomyelinase (NSMase); S-adenosyl-l-homocysteine (SAH); S-adenosyl-l-methionine (SAM); sphingomyelinase (SMase).
Conflict of interest: The authors have declared that no conflict of interest exists.
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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After outpacing the industry, private sector non-life insurance firm SBI General Insurance is aiming to break even during the current financial year, a top official said.
Compared to last year, we have grown by 39 per cent in the last six months, that is till September. (Non-life insurance) industry grew by 29 per cent during the same period, SBI General Insurance Deputy CEO, Andrew Farlow told reporters.
Asked about plans for the current financial year, he said We had reported first time profits this year.
The company, during the first six months of the current financial year ending September 30, 2016, has collected a gross written premium of Rs 1,200 crore, as against Rs 2,042.50 crore registered during the last financial year.
Our focus is more on meeting the bottomline profits,he said, adding the company's 80 per cent portfolio was in retail while the rest comprise commercial and SMEs.
To a query on whether the company was eyeing Rs 2,500 GWP during the current financial year, he said, It depends. Focus is on growing the business very profitably.
SBI General Insurance is a 74:26 joint venture between country's largest public sector State Bank of India and Insurance Australia Group. It has about 7,500 agents across the country.
Curated from On track to break even in FY17: SBI General Insurance


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Sundance, the leading supplier and manufacturer of advanced digital signal processing (DSP) and reconfigurable FPGA systems, announced availability of its second generation, high performance SMT8121 communications development platform. The announcement coincides with a decision by the UK's communications services regulator Ofcom to permit the use of mobile phones on aircraft. Passengers' own mobile phone handsets will be able to connect to an on-board base station and access a range of communications services.
Already used as the environment of choice for High Speed Direct IF by some of the world's leading electronics companies, the SMT8121 delivers 1Gsps sustained performance and features high performance, low power FPGAs and DSPs connected to multi-channel high speed data converters from e2v. The SMT8121 is a modular system comprising e2v's AT84AD001BCTD Dual 8-bit 1Gsps ADC, multiple Xilinx Virtex II Pro and Virtex 4 FPGAs, TI C64 DSP and DAC technology from Fujitsu. Used for multi-carrier/ multi-standard cellular systems, high direct-IF infrastructures, radar systems and test equipment the SMT8121 provides two input and two output channels each sampling at 1Gsps.
"In the SMT8121 we have a platform that is geared to supporting an array of next generation communications services and roadmap applications for the hand-set manufactures and cellular base-station providers", said Flemming Christensen, managing director of Sundance Multiprocessor Technology Ltd. "The SMT8121 supports the drive for a Software Defined Radio platform that can handle Direct IF, and its low power and high performance mean it is a practical solution in environments that range from the boardroom to the battlefield. With plans now confirmed for airlines to offer mobile communication services we are also seeing interest from the developers of advanced in-flight communications systems."
With the SMT8121 focused on applications where fast sampling speed (up to 2Gsps), flat spectral output, high input analog bandwidth and very low power consumption are key factors, the dual channel ADC data converter from e2v technologies was selected by Sundance developers. The e2v solution not only met the GHz performance and low power specification needed for the SMT8121, but also provided a user-friendly serial programming (3-wire) interface for rapid design and set-up over a range of target markets including aerospace, defense and satellite.
"Our fast GHz ADC technology has provided a benchmark for design flexibility, ease-of-use and performance for numerous applications including software radio, test equipment and communications" said Andrew Benn, Product Marketing Manager at e2v. "e2v continues to provide technology leadership with higher performance and greater design flexibility with the Quad 8-bit 5Gsps ADC. The investment made by Sundance in our ADC technology is testament not only to the technical performance of our solutions but also the cost advantage we provide in very high performance systems."
The SMT8121 is fully supported and supplied with the 3L Diamond design environment that allows the user to easily and efficiently implement algorithms on any of the available DSPs, FPGAs and PowerPCs. Host-side software interface to the PC is included and the solution is supported by TI's Code Composer Studio.
Available immediately from Sundance, the SMT8121 provides maximum flexibility and field upgradability to a range of FPGA and DSP parts. The SMT8121 maintains the legacy tradition of Sundance's forward and backwards compatibility.
Ofcom is the independent regulator and competition authority for the UK communications industries, with responsibilities across television, radio, telecommunications and wireless communications services.
About Sundance
Sundance designs, develops, manufactures and markets internationally high performance signal processing and reconfigurable systems for original equipment manufacturers in the wireless and signal processing markets. Leveraging its multiprocessor expertise and experience, Sundance provides OEM with modular DSP and FPGA-based systems as well as data acquisition, I/O, communication and interconnectivity products that are essential to multiprocessor systems where scalability and performance are essential. With over fifty different modules and carriers for PCI, cPCI VME and Stand Alone platforms, Sundance is a solution provider to semiconductor, pharmaceutical and factory automation industries. Sundance, founded in 1989 by the current directors, is a member of the TI Third Party Network, Xilinx Xperts and MathWorks' Connection programs.
About e2v
e2v technologies is a leading designer, developer and manufacturer of specialised components and subsystems, falling within two product groups: Electronic tubes and Sensors and Semiconductors. These products enable some of the world's leading OEMs to deliver innovative systems for medical and science, aerospace and defence, and commercial and industrial applications. For the year ended 31 March 2007, e2v achieved sales of £174m and is listed on the London Stock Exchange (e2v.l). e2v has approximately 1,800 employees worldwide with three UK based manufacturing sites in Chelmsford, Lincoln and High Wycombe, and one in Grenoble, France and one in Corcelles, Switzerland. In addition e2v has sales offices in the UK, USA, Germany, France and Hong Kong, as well as a network of distributors and representatives covering other key territories.


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Hecarim Build Guide by JakanKenshin
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Hecarim Build
Ability Sequence
Not Updated For Current Season
The masteries shown here are not yet updated for the current season, the guide author needs to set up the new masteries. As such, they will be different than the masteries you see in-game.
Welcome to my guide on Hecarim, the Shadow of War. Some background on me: I have no background. I am a relatively new player and I am making these recommendations based upon my experience thus far. If you have any suggestions or pointers they are welcome. This is my first guide so I do apologize for any errors in coding or any lacking segments.
Hecarim is considered a melee fighter. Given his natural defensive abilities it is best to play him as an offtank. He is an excellent ganker and initiator. Due to my lack of expertise with jungling I will not be presenting a jungle build in this guide.
The first build I present I consider to be the most viable, it is a pretty standard build.
The second, hybrid build is the one I have the most fun playing as it gives you a large damage bonus with both Spirit of Dread and Onslaught of Shadows.
I will not be including a section on warding, jungling, farming or zoning as great guides already exist for those specifically and I have not acquired permission from the makers of any of those guides to use or reference them here.
As this is my first guide I will be tweaking as time goes on. Any feedback on what needs tweaking is more than welcome. I will primarily be focusing on fleshing out some of the sections and improving the aesthetics as my proficiency with the code increases.
LaneRim Pros
High and sustainable damage output
A well rounded mid-cost build
Very good at disrupting enemy team
LaneRim Cons
The lack of stun abilites can leave you vulnerable
Hyrbrid Hec Pros
Life steal+Spell Vamp+ Spirit of Dread = free hps, ftw
Hybrid Hec Cons
Expensive build
As I almost exclusively play tanky dps ad characters the runes selected for this build are largely chosen for economic reasons. They are all solid runes though and I usually take greater quintessence of desolation instead of Greater Quintessence of Movement Speed.
greater mark of desolation gives you some needed arpen - a very common choice
gives you a decent armor boost early game - also a typical choice
will give you scaling magic resist which will be a great help if you're stuck laning with the enemy AP champion.
will give you a slight speed boost and work with Hecarim's passive ability to give you some bonus AD.
The masteries are selected to emphasize his natural abilities as well as make up for those he lacks. No matter what build you are making I strongly recommend investing the points into Swiftness and Runic Affinity . Swiftness will build with his passive and allow him to initiate and gank faster. Given Hecarim's natural ability to jungle investing the point in Runic Affinity allows you to get the most out of the buffs offered in the jungle.
Other than that I keep the masteries pretty standard. building up to Weapon Expertise in offense and taking defense masteries to bolster health, armor, and magic resist.
The hybrid build I lean toward a glass cannon on. I focused on offensive masteries to balance the power of his abilities to deal maximum damage. The 21st point in offense is a bit hard to place, I put it in either Vampirism or Deadliness
Now the fun part.
Recommended build For LaneRim:
Starting Items
a great starting item made greater by Warpath
pick up three of these for some early sustain
a solid starting item, but no building opportunities
Early Game Items
should be your top priority as it works wonderfully with Rampage once you have your stacks.
dat tenacity.
only if they brought AD heavy composition.
Mid Game Items
for mid game allows you to build up its bonus and give you some pretty awesome sustain.
couldn't hurt.
an uncommon item, I really like its shield and damage bonus off missing health, combined with your healing powers you can become very dangerous near death.
Late Game Items
great item, and don't forget the speed bonus
turn all that tankyness into some raw power!
zeke's herald lifesteal and a nice little aura for your friends
Other viable items
grants you some MR while increasing your speed and therefore AD.
is another decent choice as it makes 's healing more effective.
Looking to stack some flat AD? I only really recommend this if your carries are not putting out enough damage or getting focused down too quickly, but it's hard to go wrong with The Bloodthirster or an Infinity Edge.
If your team is short on support you can easily swap in a Aegis of the Legion, Frozen Heart, Randuin's Omen or shurelya's reverie.
Recommended Items for Hybric Hecarim:
Starting Items
a great starting item made greater my Warpath
pick up three of these for some early sustain
a solid starting item, but no building opportunities
Early Game Items
should be your top priority as it works wonderfully with Rampage once you have your stacks.
dat tenacity.
only if they brought AD heavy composition.
get that farm going. The armor and extra AD are very helpful at lower levels, you may want to rush this as your second item.
Mid Game Items
every hybrids friend
steal you some hps
steal you some more hps, I wait to build Hextech Gunblade until after Trinity Force once I have the utility from the Vampiric Scepter and Hextech Revolver
another must for hybrids
Late Game Items
finish it off for that tasty active (which I always forget to use)
boost that attack speed, mobility and damage, courtesy of Warpath
great for shredding tanky enemies and farming/jungling.
Other viable items
For some more attack speed, arpen, AD, CDR, crit chance and an active speed (thus damage) boost go for Youmuu's Ghostblade
Wit's End is also a great choice, giving you some bonus damage and basic attacks and buffing your MR
Given the frailty of this build a Guardian Angel could turn a team fight around.
If you are getting caught in cc too much a Banshee's Veil will suffice, plus some much needed MR.
This armor-starved build could certainly benefit from a Sunfire Cape, Warpath will allow you to stay right on top of running enemies.
I don't add some common hybrid items such as Abyssal Mask and Rylai's Crystal Scepter, I feel Hecarim's true power comes from AD and those item slots would be put to better use with sustaining or more AD focused items.
Skill sequenceRampage is your primary source of damage and Spirit of Dread keeps you in lane I strongly recommend maxing these asap.
My only other suggestion would be to work on leveling Spirit of Dread faster with the Hybrid Hec build.
After investing my first point into Devastating Charge I leave it. I primarily use this skill as utility and find the increases per level of the other skills to be more useful.
Summoner Spells
I consider Flash to be indispensable. A well placed Flash can change the outcome of a team fight or deny the enemy team a kill.
Ignite and Exhaust are both great spells, especially for the hybrid hec build.
I really love Teleport on this champion. Many a time have I ported onto a turret and used Onslaught of Shadows to send the enemy straight into the tower, thinking they could tank it.
Ghost is a great choice as well, it allows you to get into position faster and increased your passive damage bonus.
Skill usage
Hecarim's skills are primarily AOE and can be absolutely game changing in a team fight. You just need to know when to use them.
is a sexy, sexy passive. Ignoring unit collision is great for chasing or running. A word of caution, minions will not stop you. This may sound stupid and obvious but I learned quickly that I use minions to control my champions movement in small ways. Such are taking down a turret - being directly behind the first minion is great as it allows you the most time to hit the turret for that minion wave. Trying to run behind the first minion on Hecarim may leave you tanking unexpected turret shots as the minions will not keep you in check.
a powerful melee aoe attack. It can gain stacks which reduce its cooldown time. This ability synergizes beautifully with Sheen, allowing you to proc with every strike. This is your primary source of damage and should be maxed first.
is what keeps you in lane. There is no cap on how much you can heal from this ability on champions. You can use this ability with Rampage to harass effectively or secure a kill. If using the hybrid build this ability can clear out minions very quickly, granting you health in the process. I max this ability second.
is similar in function to Headbutt, serving as a great initiating move. The boost in speed will increase your AD. Timing this move to get the most damage out of it can be tricky. All I can say is practice. The speed bonus also allows you to chase or flee quickly. Be sure to run just past your enemy before striking to knock them away from their allied turrets.
this is a freaking awesome ult. I fully expect a nerf to come soon. AOE fear ability that does significant damage over a large area. You can use it to dash just beyond fleeing enemies to cause them to run away from their base or use it to change the entire flow of a team fight. The hybrid build adds significant damage to this ability.
C-c-c-combo breaker!
Hecarim can harass very easily. since you can ignore unit collision with Warpath you can easily stack up and charge in. Pop Spirit of Dread just before using your stacked Rampage this will heal you from the rampage strikes, spirit of dread damage, and any basic attacks you make. Once done you can use Devastating Charge to knock the opponent back or speed away.
If committing to a kill instead use Devastating Charge to knock the enemy into an advantageous position for you and your allies, whether that be away from a turret or into an ulting Nunu or Galio.
Thank you for taking the time to look at my guide! If you have any questions or suggestions on how to improve (both as a guide maker and player) please let me know!
A huge thanks to jhoijhoi for creating the guide to creating guides, :)


text:
Tribal Color Pop Drawstring Backpack
Hit the hallways in style with this trendy backpack! It features a canvas body with colorful tribal prints and a patched elephant at the flap closure. A decorative buckle with a magnetic snap completes the look.
- Base: approx. 12.5 in. wide x 6.25 in. deep; height: approx. 15 in.
- Drawstring closure
- External pouch pockets, front with zip closure
- Fully lined
- Adjustable straps
- Man-made materials
- Imported


text:
Al Qaida suspect arrested in burka
A suspected German al Qaida member wearing a woman's burka has been detained after leaving Pakistan's militant heartland close to the Afghan border.
The 27-year-old man, allegedly an expert in making suicide vests, crossed into Pakistan from neighbouring Afghanistan a few months ago.
Police said the German was in a vehicle with a local man and his young daughter when he was picked up at a check post.
Press Association


text:
[Plugin: RSFiles! – Download Manager] How to get RSFiles to work
I figured out how to get this to install, though I am a bit shocked that the creator wouldn't figure this out for users. Simple install and getting it to work shouldn't require paid support.
Anyways on to the how-to.
The plugin zip that you download from the wpress extend site has a zip file inside a zip file, and the automatic installer can't handle it.
So do this:
Step 1: Delete the rsfiles-download-manager folder from your plugins directory if you've already tried to install it.
Step2: Download the RSFiles zip from the wpress extend site to your desktop
Step 3: Unzip the file you downloaded.
Step 4: Go to your wp-admin plugin page, click on add new, and click on the upload button to manually upload the plugin.
Step 5: Inside that file you unzipped is another zip file called rsfiles.zip Upload that zip file and it will install and activate just fine!
- The topic '[Plugin: RSFiles! – Download Manager] How to get RSFiles to work' is closed to new replies.


text:
2008 Audi A5Enlarge Photo
The Nuvolari evolves into a stunner two-door.
Preview by Marty
Padgett (3/5/2007)
New two-door spun from Arnage; 550 to be built.
2008 Jaguar XJ Preview by Marty Padgett
A more contemporary look – or a bridge to the future?
Volvo V70 Ready for Geneva Debut by TCC Team
New wagon is based on S80, gets turbo engine.
Preview by Marty Padgett
A cross-country spin on the new V70.
Benz C-Class Gets Diesel in Geneva by
Marty Padgett (2/26/2007)
New BLUETEC needs no additives, Benz says.
Lambo Superleggera to Geneva by Henny Hemmes
Lightweight Gallardo debuts at 2007 show.
Maserati Gran Turismo Wows Geneva by Richard
Yarrow (3/6/2007)
Daring styling, revamped mechanicals for 2+2.
Preview by Henny Hemmes
The middle child gets some loving attention.
XKR Gets Portfolio Treatment by Marty
Padgett (3/6/2007)
Styling cues, 20-inchers tune up Jag's look.
Opel Gets OPC, Hottest Corsa Ever by Richard
Yarrow (3/6/2007)
OPC drops 60 in 6.8 seconds. Jealous much?
MINI One, Diesel Motor to Geneva by Marty
Padgett (3/7/2007)
Newest editions not likely for U.S.
Hyundai i30 Hits Geneva Stage by Richard
Yarrow (3/7/2007)
New hatchback is Focus-sized, shaped.
Mazda2 Takes a Geneva Bow by Marty Padgett
Subcompact could be coming to the States.
Ford–and 007–Present New Mondeo by Henny
Hemmes (2/13/2007)
New sedan spins off wagon seen in Casino Royale.
2007 Skoda Fabia Supermini Grows by Richard
Yarrow (12/12/2006)
A bigger minicar? Right next to the jumbo shrimp.
Fiat Abarth Returns at Geneva by Richard
Yarrow (3/7/2007)
Famous name applied to new Punto hatchback.
Fiat Bravo, Abarth Meet in Geneva by Richard
Yarrow (3/7/2007)
Scorpion badge hits Punto; 500 is next.
Daihatsu Plays Scion xB Again by Henny
Hemmes (3/6/2007)
No Euro Scions, but Materia materializes.
Nissan Takes to the X-Trail by Henny
Hemmes (3/6/2007)
More giddyup for Nissan's non-U.S. ute.
Time for Tatas – Ten Years of Them by Henny
Hemmes (3/6/2007)
Indian car company gets Elegante for Geneva.
Renault Revisits the Twingo by Henny Hemmes
An icon goes under the knife.
BRABUS smarts Scoot to Geneva by Marty Padgett
Second-gen smart gets power, styling mods.
Geneva Microbrands: Gumpert, Tramontana by
Richard Yarrow (2/27/2007)
Sportscars duel to Geneva, 60 mph.
2007 Dodge Demon conceptEnlarge Photo
Carbon fiber roof, "typical" V-8 give away real coupe.
Will Dodge Deliver the Demon? by TCC Team (3/6/2007)
Roadster concept appears at Chrysler turning point.
Hyundai Has Good QarmaQ by Richard Yarrow
Geneva concept is plasticky, intentionally.
Opel GTC: A New Design Era? by Marty Padgett
Gran Turismo – no, not that one – rides the runway.
by Marty Padgett
A "nimble" roadster for Dodge's Geneva show stand.
2007 Rinspeed eXasis Concept by Marty Padgett
See-through publicity: those wacky Swiss go see-through for "3-D" concept.
Mazda Hakaze Concept by Marty Padgett (3/7/2007)
A trio is completed with a Geneva concept.
2007 Honda Small Hybrid Sports Concept by
Henny Hemmes (3/7/2007)
Honda bringing hybrids, diesels to Geneva.
Brand's first convertible comes to Swiss show.


text:
I've been here three times and ever ytime I had a wonderful time here.
The staff is very friendly and helps you with everything. They can arrange transport to the main village of Phangan if you wish.
The pool is excellent (includes a pool bar) and lies next to the beach/restaurant. The beach is perfect. Very quiet bay with a wonderful view. The restaurant serves basic food which is good enough but not the best. There are several other restaurants if you take a walk down the beach.
The rooms are very basic. Since you only sleep here, I don't really mind. The bathrooms are clean.
Only bad thing I could come up with is the location. If you're attending the Full Moon Party, you have to make sure that you're not too drunk on your way there or on the way back. The ride is very bumpy and takes about 40 minutes. At night, this road isn't lit, so I wouldn't suggest renting a bike.
- Reservation Options:
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- Also Known As:
- Koh Phangan Dreamland Hotel Koh Phangan


text:
Each button in the Admin bar has a self-defining and space-saving icon. But the link to the blog frontend (which is represented as a blog title), has no icon. If you want, you can change it quickly – here is a small solution.
The following code belongs in the
functions.php of the Theme, or better, in a Plugin. It adds the current favicon before the blog title. And to fit the image into the overall picture of the admin bar, we darkened it and and gave it a round shape.
* Plugin Name: Icon for Admin Bar Site Link
* Plugin URI: http://wpengineer.com/?p=2366
* Description: Add Icon to Site Link in the WordPress Admin Bar
* Version: 1.0.0
* Author: Sergej Müller
* Author URI: http://ebiene.de* License: GPLv3
// This file is not called from WordPress. We don't like that.
! defined( 'ABSPATH' ) and exit;
// If the function exists this file is called as comments template.
// We don't do anything then.
if ( ! function_exists( 'sm_add_adminbar_site_icon' ) ) {
// add to admin area, inside head
add_action( 'admin_head', 'sm_add_adminbar_site_icon' );
// add to frontend, inside head
add_action( 'wp_head', 'sm_add_adminbar_site_icon' );
function sm_add_adminbar_site_icon() {
if ( ! is_admin_bar_showing() ) {
echo '<style>
#wp-admin-bar-site-name > a.ab-item:before {
float: left;
width: 16px;
height: 16px;
margin: 5px 5px 0 -1px;
display: block;
content: "";
opacity: 0.4;
background: #000 url("http://www.google.com/s2/u/0/favicons?domain=' . parse_url( home_url(), PHP_URL_HOST ). '");
border-radius: 16px;
#wp-admin-bar-site-name:hover > a.ab-item:before {
opacity: 1;
Guest Post
This post is written by Sergej Müller wpseo.de, who created the popular WordPress Plugin to optimize your SEO.
Thank you very much from our part to Sergej.
If you also like to have your interesting post published on our website, please let us know on our contact page. Of course we will appreciate your contribution!


text:
Have been going to Bert's for the past 5 yrs,
""I warn you to go elsewhere.
Pros: it was sunny that day.
Cons: Cost overrun without warning. Sloppy work. Rude owner. Messy work.
From family friendly trips with kid friendly activities to local plumbers lawyers, spas, and contractors. Judy's Book has millions of listings and reviews for the best and not so great. Whether you're looking for a specific business or just trying to discover great places we make it easy. We're a family oriented site focusing on bringing trusted reviews and recommendations to moms and others. Green and Sustainable places are becoming more relevant to Judy's Book users, have a favorite place? Write a review and tell users why it's Green!


text:
Life sized robot dalek prop parts
Sold for:
Start Free Trial
or Sign In to see what it's worth.
- Sold Date:
- Source: eBay UK
Middle section to make a life sized robot prop. Plus three fiberglass neck rings.
These are fiberglass robot parts and are untrimmed, unfinished. It comes with the ring parts shown in the second picture, all untrimmed.
There are some other bits around so please ask if you're interested. Please message me if you want the individual parts then I'll list them as a buy it now for you.


text: i've got a 2006 6.6L Duramax GMC 2500HD, just wondering about putting a chip or some other type of performance parts on the truck, and what type of improvement im actually looking at. seeing whats out there and whats the best move if any. the truck is already powerful enough for work, but it would just be fun to have a lot more torque and horsepower just for bragging rights, you know, my truck is bigger than yours, so i can drag some fords sideways. anybody know how many pounds of boost the turbo in a duramax pushes? i know this cant be good for the trucks, and i dont really care. over the summer i chained my gas 2500hd up to a gas ford f250, i didnt know this at the time, but turns out the ford was in 4 low and i was only in 4 high and i dragged him across the lot. that was a good time, damn was he embarrassed. havent gotten a chance to chain the duramax up to a powerstroke yet. nobody i know with a powerstroke is willing to do it. thanks


text:
You must post in this thread if your left hand is touching your face.
You must post in this thread if your left hand is touching your face.
My left hand is touching my toy, whats it called then? :))
Multitasking. Or you're waiting for your porn videos to buffer.
You got me Dando :)) and right hand on my mouse :)
I feel violated. Like I was being watched all along.
Has anyone really been far even as decided to use even go want to do look more like?
@Flash Mob,
You've got to be kidding me. I've been further even more decided to use even go need to do look more as anyone can. Can you really be far even as decided half as much to use go wish for that? My guess is that when one really been far even as decided once to use even go want, it is then that he has really been far even as decided to use even go want to do look more like. It's just common sense.
What what, in the butt
I said what what, in the butt
You wanna do it in my butt, in my butt?
Jon Gu, where have you been?
And what's so special about your butt??
This is officially the greatest thread title in WLIB history.
jesus Dando christ that was too funny.
We can let Samwell or Butters explain Jon's reference:
FYI, while google searching for those vid links, I came across this sophisticated and worthy post:
A new report published by New York City's Department of Health discovered that, while over 100,000 NYC women are having anal intercourse each year, many are not protecting themselves from HIV or other STD's...Unprotected anal sex is most common among younger women and those with multiple partners. Women 18 to 24 years old are nearly six times more likely than those aged 45 to 64 to report unprotected anal sex (11% versus 2%).
Everyone on WLIB who has had unprotected anal sex with a woman aged 45 to 64, raise your hands now. Dando is assumed.
you guys make me feel like it's so good to finally be back in beijing..
i enjoy anal sex very much.done it with 2 girls.it was fantastic.doggy style is awesome.anyone interested?we will take it slow and easy.
uggh. must wash my eyes with bleach now.
And another thread is spoiled. Thank you, Steve, for elaborating on the finer points of GOD YOU ARE AN IDIOT.
It's all I can take some days.
Not too shocked by that article though. Buttsecks is pretty much mainstream in the States these days, I guess.
I suspect a large portion of the 18 to 24 demographic comes from a situation like this:
Drunk guy: DURRR let's do it!
Drunk chic: uhhhh got no condom here tho...
Drunk guy: s'ok, i can put it in yr butt - yr drunk enough to think that's a safe and viable alternative!
Drunk chic: hehe ok!
@ Steve,
Yo dawg, I heard you like butt fuck, so we put anal into your anal so you can anal while you fuck off and don't spoil again.
Can anal sex generate any orgasm? I mean those being fucked, esp. on the woman side?
...Sounds stupid, but just out of curiosity...
And don't tell to have a try...
I've known a couple of chicks who claimed to get off from taking one up the pooper, but I remain skeptical. No prostate. But I read that in some cases clitoral nerves extend throughout the labia, the perineum, and around the anus.
A true gentleman would give a reach-around, anyway.
That's the least you can do.
And even if you don't get AIDS from cruising the hershey highway without fresh tires, it's nonetheless a good way to get an unspecified urinary tract infection. Butts are full of poop and bacteria! Wrap it up, yo.


text:
OCEAN: It's been a great week of fishing with plenty of assorted species being caught. Offshore catches out of the Hatteras marina's primarily consisted of high numbers of all size dolphin. Billfish action was very good with sailfish, blue & white marlin, and one swordfish reported. The blackfin tuna bite has backed off a bit but a few citation one's are still being caught along with a few scattered yellowfin tuna and wahoo. Nearshore anglers scored bigtime with the arrival of cobia, citation size specimens were caught on a daily basis along with plenty of spanish mackerel and moderate amounts of bluefish and atlantic bonito. Anglers fishing out of Oregon Inlet had favorable results also with dolphin dominating their catches as well. Billfish catches were on an upward trend with all types caught. Assorted tuna's were caught in moderate amounts including yellowfin, blackfin, and bigeye. Nearshore fisherman enjoyed limits of bluefish and spanish mackerel on most outings along with moderate amounts of cobia.
INLETS/SOUNDS/BAYS: Fishing success in these waters has finally made a significant improvement, very large sea mullet have been caught throughout the region in high amounts, spot, pigfish, pinfish, spotted seatrout and seemingly endless amounts of undersize striped bass were also reported. Anglers fishing off of the "Little Bridge" and underneath of the Washington Baum bridge caught some very large black drum, along with some decent spotted seatrout in the early morning hours.
PIERS/BEACH: Catches were fair from North to South, bluefish made up the bulk of the catch along with moderate amounts of sea mullet and puffers. anglers fishing from Avon southward continue to catch large specimens of pompano and sheepshead along with a few red drum of all sizes, a mixed bag of other species were caught in low amounts.
Brian A Melott
Recreational Port Agent
Division of Marine Fisheries
NC Department of Environmental Quality


text:
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Where's my paperwork?
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Volcanic ash FODCOM
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Through the month of July if you check into one of the V.I.P. Suites at the W South Beach, you'll get gratis some super-sexy women's pajamas from Ari Dein.
Exclusively made for the swank hotel chain, a limited quantity will also be available for purchase at W Hotels The Store at W South Beach and online at WHotelsTheStore.com for $165.
For the uninitiated, Ari Dein is a luxe label designed by Arielle Shapiro (who has appeared on The CW's "Gossip Girl") and is inspired by boutique hotel living and art deco style.
Bit of trivia: Shaprio's grandparents used to run Dein Fur Inc., which had a location on Lincoln Road.


text:
Heisenberg Hoodies
Bryan Cranston and Aaron Paul go into business together and this time it's legal…barely. Find out what happens when Julia Louis-Dreyfus becomes a client, trying to offload television's most coveted item, in this video starring three Emmy Award winners.
Tags: barely-legal-pawn, bryan-cranston, aaron-paul, julia-louis-dreyfus, emmy
Did a corrupted corporations or villains have taken over your life? Better Call Laurel! Someone is targeting your clients or your interests? Better Call Laurel! Hi! My name is Laurel Lance. Did you know that you have equal rights? Its a part of the constitution. So, if someone comes after you like supervillains or criminals come after you, come to me. They will fail this city!
Tags: arrow, agent-carter, saul, superheroes, better


text:
Happy Monday! I don't know where you are in the country, but we have a rainy week ahead of us here. Mother Nature missed the "April showers, brings May flowers" memo. So today is officialRead more
Tag: motivation
Overcoming Fears and Doubts in Your Weight loss Journey
A few years back, I took the plunge and competed in an NPC figure competition. A group of my friends were doing it together, and well..it made that decision an easy one. I knew IRead more
A Day in My Life
I have something a little different and a little more on the lighthearted side today! I'm going to take you through a day in my life! Enjoy! 5:45 AM: Alarm clock goes off Kane wakesRead more
Why I Stopped Reverse Dieting… & Then Started Again
Forewarning: This post could also be titled " Why it sucks to be lean". Long before I started dieting for my first figure show last fall I knew I was going to reverse dietRead more
Finding Your Motivation
There's one question I get asked all the time, "How do you stay motivated to work out?" In all honesty, I have never really known how to answer this question. My usual response is aRead more
What to do When You Don't Have Time to Exercise
What to do When You Don't Have Time To Exercise "I don't have time" may be the most overused excuse when it comes to working out and eating better. In today's world its no exaggeration. WeRead more
How To Start, And Stick To A Fitness Program
I remember the first time I stepped into a gym. I was a freshman in college and had NO idea what anything was let alone how to use it. I was not out of shapeRead more
Why Your Goals Fail – And How To Set Ones That Won't
Remember your new year's resolution? You know…the one where you were going to lose 10 lbs, or finally have the beach body you always wanted? How's it going with that? Have you given up? IfRead more


text:
swim. I swim. I swam. I will swim. I am swimming. I have swum. tear. I tear. I tore. I will tear. I am tearing. I have torn. walk. I walk. I walked. I will walk. I am walking. I have walked. throw. I throw. I threw. I will throw.
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I swim
I swam
I will swim
I am swimming
I have swum
I tear
I tore
I will tear
I am tearing
I have torn
I walk
I walked
I will walk
I am walking
I have walked
I throw
I threw
I will throw
I am throwing
I have thrown
I tie
I tied
I will tie
I am tying
I have tied
I whistle
I whistled
I will whistle
I am whistling
I have whistled
I wash
I washed
I will wash
I am washing
I have washed
I wave
I waved
I will wave
I am waving
I have waved
I wrap
I wrapped
I will wrap
I am wrapping
I have wrapped
I write
I wrote
I will write
I am writing
I have written
I yawn
I yawned
I will yawn
I am yawning
I have yawned