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- Report: #1114974
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Complaint Review: GWC Warranty
GWC WarrantyPO Box 7900 Wilkes-Barre, Pennsylvania USA
GWC Warranty Deceptive and Misleading - Big Scam Wilkes-Barre Pennsylvania
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GWC Warranty is a very deceptive warranty company for used vehicles. The warranty is presented to you that it covers everything - labor and parts.
The facts are that the company will only pay a small hourly rate to a mechanic ($63/hr) and what they feel they want for the part you may need. You are sold a warranty that lists categories of items covered and are told you can take your car anywhere for service and parts at no cost to you.
When you take your car for service, the mechanic has to call PRIOR to repairs and told how much GWC will pay for the repair. Often times it doesn't even cover the cost the mechanic paid for the part! You are not informed of any deductibles or that if you purchase a 90 day warranty the "all inclusive" list you were told were covered gets smaller.
This report was posted on Ripoff Report on 01/12/2014 12:59 PM and is a permanent record located here: http://www.ripoffreport.com/r/GWC-Warranty/Wilkes-Barre-Pennsylvania-18773/GWC-Warranty-Deceptive-and-Misleading-Big-Scam-Wilkes-Barre-Pennsylvania-1114974. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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Last time I was filming in studio I made a video on how to sew a kimono that you can watch here, and I made this one! I have been on a kimono kick lately and I have worn each one of mine that I have sewn to the beach and around town regularly. They are just so easy to wear and sew! I love the bold and colorful print of this fabric and that faux suede fringe trim is just what it needed to finish off the hem. I wish I bought more at the time so I could also fringe the sleeves.
For this kimono I used our draft-it-yourself kimono pattern from the 06/2016 issue, and as soon as I saw this pattern I knew I had to make one. If you want to make one yourself, go ahead and download the pattern then watch my video here!.
It was also a treat to get this kimono photographed in studio :) I'm sure my boyfriend Julian who regularly documents my sewing creations was glad to have a little break.
Happy Sewing!

subset: commoncrawl


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I'm inspired by architecture and simple everyday things in life. I love the victorian era and 17th & 18th century. I like to put romantic and vibrant colors in my designs. I'm a bit romance and rockerish. Creating is what I do. Right not I'm designing han
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Index of content:
Volume 14, Issue 5, 01 May 1946
The Scattering of Electrons by Hydrocarbon Films14(1946); http://dx.doi.org/10.1063/1.1724137View Description Hide Description
Several formulae are derived expressing the intensity of electron scattering for a variety of orientations of hydrocarbon films on a solid surface. Intensity contour maps may be constructed from these formulae for arbitrarily chosen models. These maps are helpful for determining the structure of a film and are being prepared for future publication.
A Relation between Bond Force Constants, Bond Orders, Bond Lengths, and the Electronegativities of the Bonded Atoms14(1946); http://dx.doi.org/10.1063/1.1724138View Description Hide Description
A relation of the formhas been found to hold accurately for a large number of diatomic and simple polyatomic molecules in their ground states. (The average deviation of k calculated from k observed for seventy‐one cases is 1.84 percent.) Here k is the bond‐stretching force constant, d the bond length, N the bond order, and xA and xB are the electronegativities of the bonded atoms. If k is measured in dynes/cm×10−5 and d in Angstrom units, a and b have the values 1.67 and 0.30, respectively, for stable molecules exhibiting their normal covalencies, except those in which both bonded atoms have only one electron in the valence shell; for diatomic molecules of the alkali metals,Na2, NaK, etc., a and b are 1.180 and −0.013, respectively; for hydrides of elements having a single electron in the valence shell, 1.180 and 0.040, respectively; and for diatomic hydrides of elements having two to four electrons in the valence shell, 1.42 and 0.08, respectively. Numerous applications of the relation are made and certain exceptions are pointed out.
The Thermodynamic Properties and Potential Energy of Solid Argon. II14(1946); http://dx.doi.org/10.1063/1.1724139View Description Hide Description
In the first paper with this title, a relation was derived from the available experimental data giving the Debye characteristic temperature of solid argon as a function of a (the interatomic distance) or V (the molal volume of the solid). This relation was not consistent with the usual Grüneisen assumption, which for a small range of volumes may be written approximately —Θ−1 dΘ/dV=constant. In this paper it is shown that the Grüneisen assumption, with the constant set equal to 0.18 mole cc−1 is reasonably consistent with the experimental data. On this basis a new relation between the potential energy Ep of solid argon and a is derived. Most of the earlier conclusions are unaltered, but the present basis seems more reasonable, and, as will be shown in the following paper, avoids difficulties when application is made to liquid argon.
The Thermodynamic Properties of Liquid Argon14(1946); http://dx.doi.org/10.1063/1.1724140View Description Hide Description
The thermodynamic properties and equation‐of‐state data of liquidargon are interpreted in terms of a partition function previously derived. This partition function has a part due to vibratory motion of the atoms in the liquid and a part due to translational motion. The vibratory part contains the Debye characteristic temperature Θ for the solid, evaluated for a certain interatomic distancea 0 which appears as a parameter in the partition function. a 0 is interpreted as the average interatomic distance between nearest neighbors in the liquid, and is a function of a, the latter being defined as (2½ V/N)⅓, where V is the molal volume and N Avogadro's number. With V set equal to the molal volume of the liquid at its normal melting point, a 0 is assigned a value which appears reasonable from the atomic distribution curves obtained by x‐ray studies of the liquid. With this value of a 0, the partition function for this fixed value of V gives reasonably good values of the entropy over a large range of temperatures. A small, entirely reasonable change in the partition function improves the agreement with the experimental data. The parameters in the partition function have been evaluated for other values of V by using equation‐of‐state data. This leads to a calculation of the specific heats of saturated liquidargon over a range of temperatures. The agreement with experimental data is not exceedingly close, but is probably within experimental error. A calculation is also made of the free energies of the solid and liquid phases along the melting curve. They are found to be equal to within reasonably close limits. The application to various values of V leads to the evaluation of a 0 as a function of a. The experimental curve for (∂S/∂V) T plotted against V appears to show a change of slope near the value of V characteristic of the liquid at its normal melting point. There is also a change in slope of the curve for da 0/da vs. a, at the corresponding value of a. These facts are interpreted in terms of the atomic distribution curves, which appear to indicate a change in structure of the liquid which starts near the same value of V or a. The liquid appears to change from a distorted face‐centered structure to a distorted body‐centered structure as the volume increases. Information about the potential energy of liquidargon is obtained from the experimental data by application of the partition function. It is found that the potential energy of the liquid can be rather effectively correlated with the potential energy of the solid. Finally, certain peculiarities of the equation‐of‐state data are believed to indicate a pre‐freezing phenomenon, in which particles containing appreciable numbers of atoms and having the structure of the solid, appear in the liquid. A state of this sort can be stabilized by the entropy of mixing of the solid particles with the liquid.
Pressure Dependence of Accommodation Coefficients14(1946); http://dx.doi.org/10.1063/1.1724141View Description Hide Description
The pressure dependence of accommodation coefficients of gases on metals has been explained by assuming that the accommodation coefficient varies linearly with the fraction of the surface covered with adsorbed gas. It is further assumed that the accommodation coefficient has a negligibly small value α0 on a gas‐free surface and an asymptotic value α∞ on a saturated surface. The assumptions lead to an accommodation coefficient isotherm which reproduces the pressure dependence of 119 accommodation coefficient values for ten gases on platinum with an average absolute deviation of 1.5 percent.
Theory of the Non‐Tetrahedral Carbon Atom14(1946); http://dx.doi.org/10.1063/1.1724142View Description Hide Description
The hybridization of s and p orbitals to give non‐tetrahedral orbitals is discussed. The results are applied to cyclic hydrocarbons. For cyclopentane the theory yields an H–C–H angle of 111° 1′, and for cyclobutane an H–C–H angle of 180°.
Surfaces of Solids XVII A First‐ and a Second‐Order Phase Change in the Adsorbed Film of n‐Heptane on Graphite14(1946); http://dx.doi.org/10.1063/1.1724143View Description Hide Description
The films of n‐heptane formed by adsorption on the surface of graphite between 25° and 40°C exhibit a first‐ and a second‐order phase transition. The first‐order transition occurs between the gaseous and liquid expanded phases. The second‐order transition occurs between the liquid expanded and liquid intermediate phases, and between the gaseous and liquid intermediate phases above the critical temperature. Since the behavior of the film is the same as that observed with insoluble films on aqueous subphases, the nomenclature for the latter is used. The critical constants for the gaseous film were determined as: temperature, 31°C; area, 400A2 per molecule; and film pressure, 1.05 dyne cm−1. These values are different from those found for n‐heptane on ferric oxide, which shows that the solid as well as the gas plays an important role in the determination of the adsorption isotherm. The heat of transition for the first‐order change is estimated as 13,000±5000 cal. mole−1 at 25°C. It is found at corresponding temperatures, within this large experimental error, that the heat evolved in the first‐order transition is the same on graphite and ferric oxide.
Errata: Statistical Mechanics of Transport Processes I. General Theory14(1946); http://dx.doi.org/10.1063/1.1724144View Description Hide Description
On the Weak Associations in Water and in Aniline14(1946); http://dx.doi.org/10.1063/1.1724145View Description Hide Description
A Note on Communal Entropy. Remarks on a Paper by Henry S. Frank14(1946); http://dx.doi.org/10.1063/1.1724146View Description Hide Description
Postscript to the Foregoing Note by O. K. Rice14(1946); http://dx.doi.org/10.1063/1.1724147View Description Hide Description
The Tetrahedral P4 Molecule14(1946); http://dx.doi.org/10.1063/1.1724148View Description Hide Description
Ultra‐High Frequency Denaturation of Proteins14(1946); http://dx.doi.org/10.1063/1.1724149View Description Hide Description
The Recognition of Phase Transitions in Adsorbed Films on Solids14(1946); http://dx.doi.org/10.1063/1.1724150View Description Hide Description
Aggregational States in Adsorbed Films on Incompletely Wettable Solid Surfaces14(1946); http://dx.doi.org/10.1063/1.1724151View Description Hide Description
Sedimentation Equilibria of Polydisperse Non‐Ideal Solutes14(1946); http://dx.doi.org/10.1063/1.1724152View Description Hide Description


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Is there are way to set the number of columns in the WordPress Admin Dashboard with a function?
And then define which columns you want different widgets to be placed?
I came accross the conversation here which talks about removing widgets.
I will definitely be using that to remove widgets, but I'd also like to define where the remaining widgets are located (which column) and order too if that is possible.
I want to do this just because I have a lot of users on my backend, and I want everything to look the same for everyone.
Thanks for any help, I couldn't find any info on those topics.


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MIDDLETOWN — The mayor and Common Council members have been getting complaints about an abandoned property that's creating a Dolson Avenue eyesore for motorists entering the city from the west. But city officials say there's nothing they can do about it.
Why? The abandoned Wiedy's furniture store property is actually in the Town of Wawayanda.
Middletown Mayor Joe DeStefano says the city has gotten about a dozen complaints about the property, which he called a "rat trap, overgrown with five-foot-high weeds."
The Wiedy's lot is, indeed, overgrown and its one-time distinctive red fiberglass awning is cracked. Old steel columns have fallen onto the front lawn and the inside is strewn with debris.
The Wiedy's property is across James P. Kelly way from the QuickChek store. And although the Wiedy's property is closer to Middletown's Dolson Avenue commercial district than QuickChek is, Wiedy's is in Wawayanda and QuickChek is in Middletown.
"The (municipal boundary) line is jagged at that point," DeStefano said.
DeStefano said he has talked to Wawayanda Supervisor John Razzano about the problem.
Razzano said he agrees that the abandoned store is an eyesore. "We don't like it either," he said.
Razzano said a prospective buyer, who wants to turn the building into a storage facility, has been before the town Planning Board, but has not yet purchased the building.
Razzano said a number of buyers have expressed interest in the Wiedy's property in past years but deals have fallen though.
"We'd love to see it occupied and become a vital part of the community," Razzano said.
Location, ST | website.com


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The admission by former Liberal Democrat Party Treasurer Lord Razzall, that he was offered cash for peerages "several times a year" over a period of twelve years but failed to report any of these criminal offences threatens to take the lid back off one Westminster's most unseemly and nocuous can of worms of recent decades: cash for honours.
Chinese corruption has come under the spotlight in a big way this month. Accusations that the wife of Bo Xilai, one of China's top politicians, is behind the death of British businessman Neil Heywood have also led to allegations of the couple's corruption.
It appears the phone hacking scandal occupying the UK's news pages for the past few months may have been merely the preliminary act. The main event could well be the graft and corruption probe that now seems to be linking the Sun newspaper to numbers of Metropolitan Police officers and other public officials.


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Sammy Hagar Pays Tribute to Liza Cozad-Lauser
Sammy Hagar has shared his grief over the death of Liza Cozad-Lauser, wife of his Wabos drummer David Lauser, in a note posted at his official site.
Sammy Hagar has shared his grief over the death of Liza Cozad-Lauser, wife of his Wabos drummer David Lauser, in a note posted at his official site.
Sammy Hagar and Cheap Trick will play the opening evening of the Carolina Rebellion Festival, held May 2 and 3 at the Charlotte Motor Speedway, with Slash following on the second day.
Sammy Hagar is headed for Springfield on Nov. 23, but you can see the Red Rocker immortalized as a 'Simpsons' cast member a few days early, courtesy of this publicity shot from the Fox network.
Sammy Hagar is back with another installment of this exclusive behind-the-scenes look into his new album 'Lite Roast,' just out on Mailboat Records.
Sammy Hagar recently said he'd be open to reuniting with Van Halen if everyone could really get their act together, but make no mistake -- he hasn't forgiven Alex and Eddie Van Halen.
In today's interview we will check in with Sammy Hagar and see how he feels about Eddie and Alex Van Halen and their friendship or lack of friendship...
The soft market for new albums from veteran rock acts may have put an end to one of Sammy Hagar's many projects, but he'll never say never when it comes to reuniting with Van Halen.
Sammy Hagar has been all about spontaneity over the past few years.
Sammy Hagar has put together a music and comedy show for next week to raise money for the hurricane-devastated town of Cabo San Lucas, Mexico.
With Sammy Hagar's new 'Lite Roast' acoustic album coming out this week, we thought this would be the perfect time to take a look in the Red Rocker's personal file and dig up a few lesser-known facts about the Chickenfoot frontman.
We spoke with Sammy Hagar on the 40th anniversary of the release of Montrose's 'Paper Money.'
Sammy Hagar's not letting a little thing like a hurricane stop his birthday party.


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10 great books that will teach you about gaming history
Reading is fundamental
Hardware slips 39 percent year-over-year, gaining momentum for holidays
We sit down with ESPN's Gus Ramsey to dive deeply into the Madden NFL 13 experience
Follow our advice, and you'll be headed for the Hall of Fame in no time.
Want to know about the challenges of getting a sports game out in time for new hardware?
We sit down the the man in charge of Madden player ratings, EA's Donny Moore.
Which teams will see a big numerical jump and which will take a dip? Our crystal football reveals!
Our crack sports staff peers into the future to predict the player ratings of the NFL's biggest movers and shakers
The business of sports games is more complicated than you'd expect...
Direct, detailed, and constant feeds of information to fans has changed the way sports games are previewed - forever
Who would play whom if E3 2012 were a movie?
We spoke, they listened, and Madden will never be the same again
From rebuilding its commentary to overhauling gameplay, Madden NFL 13 is going deep
Three key Madden team members take a moment to peer into the future
We examine a year of transition for Tiburon Studios as they approach their most important NCAA and Madden games yet


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muhd-shark (n) a feces.
I walked into the bathroom and found the toilet full of mudhsarks swimming around.
od uživatele expurg8d 12. červenec 2003
To stick inappropriate things in a female's vagina. So named because Vanilla Fudge did that to a groupie with an actual mud shark.
Lucy was still not fucking satisfied, so I had to mudshark her with my asthma inhaler!
od uživatele tecumseh 04. leden 2003
White man who is particularly attracted to black women.
"Down south, we call fellows like you 'mud sharks.'"
od uživatele JRI 24. listopad 2003
Sexually challanged white British man that is attracted to a black juicy gash.
That dumb limey thinks those black chicks are top quality. He is shuch a mud shark
od uživatele Cobbs 11. květen 2006
A sexual act similar to a Cleveland Steamer in which one partner (typically male) defecates into a female partner's genital area.
I totally mudsharked that chick.
od uživatele Allahkat 13. prosinec 2004
A fag.
Leonardo DiCaprio is such a mudshark.
od uživatele Anonymous 20. červenec 2003
Pouncing gay man. "Mud" is slang for shit, combined with toothy predator = ass predator.
He is a total mudshark
od uživatele Stu 04. říjen 2002


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The long running Volkswagen Jetta aims to compete directly with other economical compacts, which means a focus on lower price and simplified equipment choices, and greater emphasis on fuel efficiency. Despite its roomy interior and grown-up looks, the Jetta sedan carries the lowest sticker price in the VW lineup.
See a side-by-side comparison of the Jetta & Civic »
The Honda Civic received a substantial redesign for the 2016 model year that gave it an all-new look, new engines, and new body styles. It's an impressive package of enhancements for a model that's still so new to the market.
How does the value-oriented Jetta stack up against the reinvigorated Civic?
Where the Jetta Delivers
The Jetta optimizes the notion of a lot of car for the money. Interior and trunk space come close to midsize levels, and features like heated outside mirrors, a 5-inch touchscreen, Bluetooth connectivity, and express up/down windows are included in the modest base price. The Jetta offers clean and sensible styling inside and out, which makes it unique in a field that thrives on trends and cuteness.
The standard 1.4-liter turbo engine makes 150 horsepower and 184 pound-feet of torque, but buyers can upgrade to a 1.8-liter with 170 horsepower, or a 2-liter turbo with 210 horses. There is also a hybrid model that cranks out 170 horsepower and gets up to 48 mpg combined.
Where the Civic Delivers
The redesigned Civic's new look puts it in a class of its own, and buyers will eventually have three body styles to choose from: sedan, coupe, or hatchback. The Civic manages to pass the well-equipped Jetta in terms of features, as it comes standard with more desirable features like a rearview camera, auto climate control, and more.
The Civic's base 2-liter engine is slightly more powerful than the Jetta at 158 horsepower, but it does fall short in torque. With its optional 1.5-liter turbo engine, the Civic pushes out 174 horses and 162 pound-feet of torque. Both of these gasoline engines easily beat the VW's 1.4- and 1.8-liter engines in fuel economy. Soon, the Civic will have an Si model to compete with the 2-liter-equipped Jetta GLI.
Which Stands Out?
The Civic's new look and new engines make it a standout in its class, leading it to a pretty easy victory over the Jetta. While the Jetta's cabin may be a little roomier than the Civic's, the differences are so small that they are not enough to overcome all the good things the Civic does.
Our Verdict: Honda Civic
A new look and new engines make the Civic our choice.
Take a closer look at the Volkswagen Jetta »


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Andrew has been performing for audiences in the Detroit Metropolitan area and beyond for nearly 40 years. He began singing in area community theatre groups at the age of 12, singing roles in "The Music Man", "The Sound of Music" and "Gypsy". Later, Andrew returned to the stage in local productions as Sid in "The Pajama Game", Tony in "West Side Story" and Curly in "Oklahoma". Most recently, Andrew appeared as The Singing Soldier in "The Living Stations of The Cross", earning excellent reviews from WJR, Catholic Radio, and The Michigan Catholic. Andrew has toured Europe 5 times as a soloist and in various groups. Andrew attended Wayne State University as a Board of Governor's Talent Scholar, earning a Bachelor of Music in Vocal Music/Performance in 1988.
Andrew has been on the staff of St. Regis Catholic Church in Bloomfield Hills since March of 1989, serving as Minister of Music, Choir Director, and Cantor along the way. Andrew also serves as Cantor at the Archdiocesan level for special events, and is the Vice President/Membership Chair for the National Association of Pastoral Musicians, Detroit Chapter. Andrew lives in Clinton Township, MI with his three children.
All are welcome at St. Regis! Please come and join us for Mass. We celebrate Mass every Saturday at 4:30 p.m. and Sunday at 8:00 a.m., 10:00 a.m., 12 noon and 5:30 p.m. We also celebrate Mass Monday - Friday at 6:45 and 8:30 a.m. and Saturday at 8:30 a.m. We would love to see you there!


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Human N-acetyltransferase 1 (NAT1) *10 and *11 alleles increase protein expression via distinct mechanisms and associate with sulfamethoxazole-induced hypersensitivity
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Pharmacogenetics and genomics
N-acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens. NAT1 alleles *10 and *11 have been proposed to alter protein level or enzyme activity compared to wild-type NAT1 *4 and to confer cancer risk, via uncertain pathways. This study characterizes regulatory polymorphisms and underlying mechanisms of NAT1 expression.
We measured allelic NAT1 mRNA expression and translation, as a function of multiple transcription start sites, alternative splicing, and three 3′-polyadenylation sites in human livers (one of which discovered in this study), B lymphocytes, and transfected cells. In a clinical study of 469 HIV/AIDS patients treated with the NAT1/NAT2 substrate sulfamethoxazole (SMX), associations were tested between SMX induced hypersensitivity and NAT1 *10 and *11 genotypes, together with known NAT2 polymorphisms.
NAT1*10 and *11 were determined to act as common regulatory alleles accounting for most NAT1 expression variability, both leading to increased translation into active protein. NAT1*11 (2.4% minor allele frequency) affected 3′polyadenylation site usage, thereby increasing formation of NAT1 mRNA with intermediate length 3′UTR (major isoform) at the expense of the short isoform, resulting in more efficient protein translation. NAT1 *10 (19% minor allele frequency) increased translation efficiency without affecting 3′-UTR polyadenylation site usage. Livers and B-lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 immunoreactivity and NAT1 enzyme activity than the reference genotype *4/*4. Patients who carry *10/*10 and *11/*4 ('fast NAT1 acetylators') were less likely to develop hypersensitivity to SMX, but this was observed only in subjects also carrying a slow NAT2 acetylator genotype.
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Diaz-Mitoma, F
Clinical and Diagnostic Laboratory Immunology
Antibody- and cell-mediated responses to sulfamethoxazole (SMX) were analyzed in AIDS patients with or without a history of hypersensitivity and in negative controls. In 20 of 20 (P < 0.01) human immunodeficiency virus (HIV)-seropositive patients with skin reactions to cotrimoxazole, we found SMX-specific antibodies, while only 9 of 20 and 17 of 20 HIV-seropositive patients without a history of hypersensitivity to cotrimoxazole had SMX-specific immunoglobulin M (IgM) and IgG, respectively. The levels of specific IgM and IgG were higher in patients with skin reactions than in patients without reactions (IgM, 1.0 +/- 0.19 versus 0.47 +/- 0.23 [P < 0.001]; IgG, 0.68 +/- 0.15 versus 0.47 +/- 0.14 [P < 0.001] [mean optical density values +/- standard deviations]). Seronegative controls with no history of exposure to sulfa compounds did not have SMX-specific IgG or IgM antibodies, and controls with a history of intake of SMX with or without reactions had low levels of IgG and IgM. The SMX-specific IgG subclasses were exclusively IgG1 and IgG3. None of the patients had detectable SMX-specific IgE or IgA antibodies nor did they exhibit a cell-mediated response as measured by a lymphocyte proliferation assay. Antibodies to SMX recognized N-acetyl-sulfonamide, N-(2-thiazolyl)-sulfanilamide, sulfadiazine, and sulfisoxazole but did not recognize sulfanilamide or 3-amino-5-methyl isoxazole in an inhibition assay. It is not known whether the SMX-specific antibodies associated with hypersensitivity reactions to SMX in HIV-seropositive patients have a pathogenic role in these reactions. Sulfanilamide or 3-amino-5-methyl isoxazole, on the other hand, could be potential alternative therapies in HIV-seropositive patients with a history of skin reactions to SMX.
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Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-κB and AP-1
Yang, Heping
Magilnick, Nathaniel
Lee, Candy
Kalmaz, Denise
Ou, Xiaopeng
Chan, Jefferson Y.
Lu, Shelly C.
Molecular and Cellular Biology
Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tert-Butylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidant-responsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-κB family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-κB binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-κB family members.
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Multicenter Study of Trimethoprim/Sulfamethoxazole-Related Hepatotoxicity: Incidence and Associated Factors among HIV-Infected Patients Treated for Pneumocystis jirovecii Pneumonia
Yang, Jen-Jia
Huang, Chung-Hao
Liu, Chun-Eng
Tang, Hung-Jen
Yang, Chia-Jui
Lee, Yi-Chien
Lee, Kuan-Yeh
Tsai, Mao-Song
Lin, Shu-Wen
Chen, Yen-Hsu
Lu, Po-Liang
Hung, Chien-Ching
PLoS ONE
The incidence of hepatotoxicity related to trimethoprim/sulfamethoxazole (TMP/SMX) administered at a therapeutic dose may vary among study populations of different ethnicities and hepatotoxic metabolites of TMP/SMX may be decreased by drug-drug interaction with fluconazole. We aimed to investigate the incidence of hepatotoxicity and the role of concomitant use of fluconazole in HIV-infected patients receiving TMP/SMX for Pneumocystis jirovecii pneumonia. We reviewed medical records to collect clinical characteristics and laboratory data of HIV-infected patients who received TMP/SMX for treatment of P. jirovecii pneumonia at 6 hospitals around Taiwan between September 2009 and February 2013. Hepatotoxicity was defined as 2-fold or greater increase of aminotransferase or total bilirubin level from baselines. Roussel UCLAF Causality Assessment Method (RUCAM) was used to analyze the causality of drug-induced liver injuries. NAT1 and NAT2 acetylator types were determined with the use of polymerase-chain-reaction (PCR) restriction fragment length polymorphism to differentiate common single-nucleotide polymorphisms (SNPs) predictive of the acetylator phenotypes in a subgroup of patients. During the study period, 286 courses of TMP/SMX treatment administered to 284 patients were analyzed. One hundred and fifty-two patients (53.1%) developed hepatotoxicity, and TMP/SMX was considered causative in 47 (16.4%) who had a RUCAM score of 6 or greater. In multivariate analysis, concomitant use of fluconazole for candidiasis was the only factor associated with reduced risk for hepatotoxicity (adjusted odds ratio, 0.372; 95% confidence interval, 0.145–0.957), while serostatus of hepatitis B or C virus, NAT1 and NAT2 acetylator types, or receipt of combination antiretroviral therapy was not. The incidence of hepatotoxicity decreased with an increasing daily dose of fluconazole up to 4.0 mg/kg. We conclude that the incidence of TMP/SMX-related hepatotoxicity was 16.4% in HIV-infected Taiwanese patients who received TMP/SMX for pneumocystosis. Concomitant use of fluconazole was associated with decreased risk for TMP/SMX-related hepatotoxicity.
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Variants in the Glutamate-Cysteine-Ligase Gene Are Associated with Cystic Fibrosis Lung Disease
McKone, Edward F.
Shao, Jing
Frangolias, Daisy D.
Keener, Cassie L.
Shephard, Cynthia A.
Farin, Federico M.
Tonelli, Mark R.
Pare, Peter D.
Sandford, Andrew J.
Aitken, Moira L.
Kavanagh, Terrance J.
American Journal of Respiratory and Critical Care Medicine
Background: Chronic progressive lung disease is the most serious complication of cystic fibrosis (CF). Glutathione plays an important role in the protection of the CF lung against oxidant-induced lung injury.
Objectives: We hypothesized that a polymorphism in a novel candidate gene that regulates glutathione synthesis might influence CF lung disease.
Methods: In a cross-sectional study, subjects were recruited from CF clinics in Seattle and multiple centers in Canada. We tested for an association between CF lung disease and a functional polymorphism in the glutamate-cysteine ligase catalytic subunit (GCLC) gene. Multiple linear regression was used to test for association between polymorphisms of GCLC and severity of CF lung disease while adjusting for age, Pseudomonas aeruginosa infection, and cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Analysis was repeated for patients with CF stratified by CFTR genotype.
Measurements and Main Results: A total of 440 subjects with CF participated in the study (51% male; mean [± SD] age, 26 ± 11 yr; mean FEV1, 62 ± 28% predicted). In the total population, there was a trend toward an association between GCLC genotypes and CF lung disease (linear regression coefficient [SEM], 1.68 [1.0]; p = 0.097). In the stratified analysis, there was a highly significant association between GCLC genotype and CF lung function in subjects with a milder CFTR genotype (linear regression coefficient [SEM], 5.5 (1.7); p = 0.001).
Conclusions: In patients with CF with a milder CFTR genotype, there is a strong association between functional polymorphisms of the GCLC gene and CF lung disease severity.
CFTR genotype; glutathione; modifier genes
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Overexpression of glutamate–cysteine ligase protects human COV434 granulosa tumour cells against oxidative and γ-radiation-induced cell death
Cortes-Wanstreet, Mabel M.
Giedzinski, Erich
Limoli, Charles L.
Luderer, Ulrike
Ionizing radiation is toxic to ovarian follicles and can cause infertility. Generation of reactive oxygen species (ROS) has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione (GSH) sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of GSH is protective. The rate-limiting reaction in GSH biosynthesis is catalysed by glutamate–cysteine ligase (GCL), which consists of a catalytic subunit (GCLC) and a regulatory subunit (GCLM). We hypothesized that overexpression of Gclc or Gclm to increase GSH synthesis would protect granulosa cells against oxidant- and radiation-induced cell death. The COV434 line of human granulosa tumour cells was stably transfected with vectors designed for the constitutive expression of Gclc, Gclm, both Gclc and Gclm or empty vector. GCL protein and enzymatic activity and total GSH levels were significantly increased in the GCL subunit-transfected cells. GCL-transfected cells were resistant to cell killing by treatment with hydrogen peroxide compared to control cells. Cell viability declined less in all the GCL subunit-transfected cell lines 1–8 h after 0.5 mM hydrogen peroxide treatment than in control cells. We next examined the effects of GCL overexpression on responses to ionizing radiation. ROS were measured using a redox-sensitive fluorogenic dye in cells irradiated with 0, 1 or 5 Gy of γ-rays. There was a dose-dependent increase in ROS within 30 min in all cell lines, an effect that was significantly attenuated in Gcl-transfected cells. Apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and activated caspase-3 immunoblotting, was significantly decreased in irradiated Gclc-transfected cells compared to irradiated control cells. Suppression of GSH synthesis in Gclc-transfected cells reversed resistance to radiation. These findings show that overexpression of GCL in granulosa cells can augment GSH synthesis and ameliorate various sequelae associated with exposure to oxidative stress and irradiation.
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Serum Peak Sulfamethoxazole Concentrations Demonstrate Difficulty in Achieving a Target Range: A Retrospective Cohort Study
Dao, Bao D.
Barreto, Jason N.
Wolf, Robert C.
Dierkhising, Ross A.
Plevak, Matthew F.
Tosh, Pritish K.
Current Therapeutic Research, Clinical and Experimental
Trimethoprim (TMP)/sulfamethoxazole (SMX) has consistently demonstrated great interindividual variability. Therapeutic drug monitoring may be used to optimize dosing. Optimal peak SMX concentration has been proposed as 100 to 150 μg/mL. The objective of our work was to determine the success rate of a TMP/SMX dosing guideline in achieving a targeted serum peak SMX concentration range.
Our retrospective cohort study enrolled 305 adult hospitalized patients who received treatment with TMP/SMX and underwent serum peak SMX concentration monitoring from January 2003 to November 2011. Patients receiving low-dose TMP/SMX therapy (TMP <15 mg/kg/d) were compared with those receiving high-dose therapy (TMP >15 mg/kg/d).
Patients were classified into peak and modified peak SMX concentration cohorts based on time between TMP/SMX dose and SMX quantification. The association between dosing group and the outcome of the SMX level within the goal range was measured using logistic regression models. The primary outcome measured was serum peak SMX concentration 100 to 150 μg/mL. Serum peak SMX concentrations were attained within range for the peak and modified peak cohort 29% and 26% of the time, respectively. The median peak SMX concentration was 144 μg/mL (range 25–471 μg/mL). The low daily dose cohort demonstrated a trend toward improvement in the odds of target peak concentration range attainment. The results were similar regardless of the method used to adjust for baseline characteristics. The pure peak and modified peak cohorts had 44% and 46% of patients with above-target SMX peak concentrations, respectively.
Attainment of the intended target concentration range was low with no difference in attainment between the low-dose and high-dose cohorts. Higher proportions of patients had an above-target SMX peak, which may indicate that the dosing algorithm is overly aggressive in obtaining the therapeutic goal.
Serum peak concentration; Sulfamethoxazole; Therapeutic drug monitoring; Trimethoprim
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Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine
Bhusari, Sachin
Abouraya, Mahmoud
Padilla, Marcia L.
Pinkerton, Marie E.
Drescher, Nicholas J.
Sacco, James C.
Trepanier, Lauren A.
Archives of toxicology
Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b5 (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity towards SMX-NO, and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue drug-adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b5, as well as significant decreases in SMX-HA reduction in SMX-NO treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in AIDS patients with antioxidant depletion.
Guinea pig; antioxidants; drug hypersensitivity; NADH hydroxylamine reductase
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Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity
Backos, Donald S.
Brocker, Chad N.
Franklin, Christopher C.
Toxicology and applied pharmacology
The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H2O2-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.
glutathione; glutamate cysteine ligase; GCLC; GCLM; TAT; protein transduction
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Transcriptional Regulation of Rat γ-Glutamate Cysteine Ligase Catalytic Subunit Gene is Mediated through a Distal Antioxidant Response Element
Shenvi, Swapna V.
Smith, Eric J.
Hagen, Tory M.
Pharmacological research : the official journal of the Italian Pharmacological Society
Despite it being a quintessential Phase II detoxification gene, the transcriptional regulation of the rat γ-glutamate cysteine ligase catalytic subunit (GCLC) is controversial. Computer-based sequence analysis identified three putative antioxidant response elements (AREs) at positions −889 to −865 (ARE1), −3170 to −3146 (ARE2) and −3901 to −3877 (ARE3) in the 5'-flanking region of the transcriptional start site. Transfections of individual ARE-luciferase reporter gene constructs into H4IIE cells, a rat hepatoma cell line, identified ARE3 as the functional promoter. Chromatin immunoprecipitation assays using primary rat hepatocytes showed that the transcription factor Nrf2, which is known to regulate ARE-mediated genes, is associated with ARE3. Co-transfection of H4IIE cells with luciferase reporter plasmids containing Gclc ARE3 and an Nrf2 expression plasmid resulted in a 3-fold activation of ARE3-mediated transcription relative to controls. "Loss-of-function" analysis for Nrf2 by small interfering RNA (siRNA) revealed that ARE3-mediated expression was significantly impaired while site-directed mutagenesis of the ARE3-luciferase reporter abolished Nrf2-mediated induction. Treatment with two known Nrf2 inducers, R-(α)-lipoic acid and anetholedithiolethione, showed that the inducible expression of the GCLC gene was also regulated by the ARE3 element. Taken together, these results show that Nrf2 regulates the constitutive expression of rat Gclc through a distal ARE present in its 5'-flanking region. This is the first report showing that rat Gclc is under the transcriptional control of the Nrf2-ARE pathway on a constitutive basis.
NF-E2-related factor 2; glutathione, Chromatin immunoprecipitation; detoxification; anetholedithiolethione; R-(α)-lipoic acid; Phase II detoxification
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Distinct Nrf1/2-independent mechanisms mediate As3+-induced glutamate-cysteine ligase subunit gene expression in murine hepatocytes
Thompson, James A.
White, Collin C.
Cox, David P.
Chan, Jefferson Y.
Kavanagh, Terrance J.
Fausto, Nelson
Franklin, Christopher C.
Free radical biology & medicine
Trivalent arsenite (As3+) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and cytotoxic effects of As3+. Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As3+ coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation and activity. As3+ increased the rate of transcription of both the Gclm and Gclc genes and induced the post-transcriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As3+-induced Gclc expression and attenuated induction of Gclm. As3+ induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and post-transcriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As3+ and highlight the potential importance of the GSH antioxidant defense system in regulating As3+-induced responses in hepatocytes.
arsenite; arsenic; glutamate cysteine ligase; GCL; GCLC; GCLM; glutathione; GSH; hepatocyte; Nrf2; gene transcription
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Activation of Promoter Activity of the Catalytic Subunit of γ-Glutamylcysteine Ligase (GCL) in Brain Endothelial Cells by Insulin Requires Antioxidant Response Element 4 and Altered Glycemic Status: Implication for GCL expression and GSH synthesis
Langston, William
Li, Wei
Harrison, Lynn
Aw, Tak Yee
Free radical biology & medicine
Our recent finding that insulin increased the expression of glutamate cysteine ligase catalytic subunit (GCLc) and coincident increases in GCL activity and cellular glutathione (GSH) in human brain microvascular endothelial cells (IHECs) suggests a role for insulin in vascular GSH maintenance. Here, using IHECs stably transfected with promoter-luciferase reporter vectors, we found that insulin increased GCLc promoter activity, which required a prerequisite increase or decrease in media glucose. An intact antioxidant response element-4 (ARE4) was essential for promoter activation that was attenuated by inhibitors of PI3kinase/Akt/mTOR signaling. Interestingly, only at low glucose conditions did promoter activation correlated with increased GCLc expression and GSH synthesis. Low tert-butyl hydroperoxide (tBH) concentrations similarly mediated promoter activation, but the maximal activation dose was decreased 10-fold by insulin. Insulin-tBH co-administration abrogated the low or high glucose requirement for promoter activation, suggesting possible ROS involvement. ROS production was elevated at low glucose without or with insulin; however GSH increases were not inhibited by tempol, suggesting that ROS did not or achieve the threshold for driving GCLc promoter activation and de novo GSH synthesis. The minor effect of pyruvate also ruled out a major role for hypoglycemia (± insulin)-induced metabolic stress on GSH induction under these conditions.
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Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2
Aleksunes, Lauren M.
Slitt, Angela L.
Maher, Jonathan M.
Augustine, Lisa M.
Goedken, Michael
Chan, Jefferson Y.
Cherrington, Nathan J.
Klaassen, Curtis D.
Manautou, José E.
Toxicology and applied pharmacology
The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity, and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1–4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2-dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 hrs after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP, but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.
Nuclear factor-E2-related factor 2; Nrf2; acetaminophen; APAP; hepatotoxicity; multidrug resistance-associated proteins; Mrp3; Mrp4
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Transcription Factor Nrf2-Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy
Zhong, Qing
Mishra, Manish
Kowluru, Renu A.
Investigative Ophthalmology & Visual Science
Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2–related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy.
Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy.
Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA.
Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
antioxidant defense; diabetic retinopathy; Nrf2
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Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits
Willis, Melanie Neely
Liu, Yilin
Biterova, Ekaterina I.
Simpson, Melanie A.
Kim, Heejeong
Lee, Jaekwon
Barycki, Joseph J.
Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insights into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to wild-type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using an S. cerevisiae model system. Mutant GCLC alone could not complement a glutathione-deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.
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Homocysteine stimulates antioxidant response element-mediated expression of glutamate-cysteine ligase in mouse macrophages
Bea, Florian
Hudson, Francesca N.
Neff-LaFord, Haley
White, Collin C.
Kavanagh, Terrance J.
Kreuzer, Jörg
Preusch, Michael R.
Blessing, Erwin
Katus, Hugo A.
Rosenfeld, Michael E.
Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Uptake of homocysteine induces oxidative stress in macrophages. Antioxidant response elements (AREs) are regulatory elements within promoters of genes, which protect cells against oxidative stress. The current study investigated whether homocysteine induces transcription of glutamate-cysteine ligase (Gcl), via ARE driven gene expression in mouse macrophages. Gcl is the rate-limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant. Gcl is heterodimeric and the genes encoding the subunits of Gcl contain several AREs within their 5′-promoter regions. Treatment of mouse macrophages with d-/l-homocysteine (50 µM) induced depletion of intracellular glutathione and a compensatory increase in Gcl activity. Electro mobiliy shift assays demonstrated increased binding of nuclear proteins to ARE-containing oligonucleotides. Real-time RT-PCR revealed increased mRNA-expression of the catalytic subunit of Gcl (Gclc) after treatment with homocysteine, and this occurred via increased transcription as demonstrated with luciferase promoter reporter constructs for Gclc. Additional site directed mutagenesis demonstrated that ARE4 plays a direct role in mediating induction of Gclc by homocysteine. Supershift analysis and Western blotting revealed that Nrf2 signalling is critical in homocysteine-induced activation of ARE4. Inhibition of MAP kinase activity reduced binding of nuclear proteins to the AREs, nuclear expression of Nrf2 and mRNA expression of Gclc. Western blotting demonstrated phosporylation of ERK1/2 in homocysteine treated macrophages. These data suggest that ARE-driven gene expression of Gclc via a MEK/Nrf2 pathway could help to protect macrophages from oxidative stress due to hyperhomocysteinemia.
Homocysteine; Atherosclerosis; Oxidative stress; Glutamate-cysteine ligase; Antioxidant response element
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Fucoxanthin Enhances the Level of Reduced Glutathione via the Nrf2-Mediated Pathway in Human Keratinocytes
Zheng, Jian
Piao, Mei Jing
Kim, Ki Cheon
Yao, Cheng Wen
Cha, Ji Won
Hyun, Jin Won
Marine Drugs
Fucoxanthin, a natural carotenoid, is abundant in seaweed with antioxidant properties. This study investigated the role of fucoxanthin in the induction of antioxidant enzymes involved in the synthesis of reduced glutathione (GSH), synthesized by glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS), via Akt/nuclear factor-erythroid 2-related (Nrf2) pathway in human keratinocytes (HaCaT) and elucidated the underlying mechanism. Fucoxanthin treatment increased the mRNA and protein levels of GCLC and GSS in HaCaT cells. In addition, fucoxanthin treatment promoted the nuclear translocation and phosphorylation of Nrf2, a transcription factor for the genes encoding GCLC and GSS. Chromatin immune-precipitation and luciferase reporter gene assays revealed that fucoxanthin treatment increased the binding of Nrf2 to the antioxidant response element (ARE) sequence and transcriptional activity of Nrf2. Fucoxanthin treatment increased phosphorylation of Akt (active form), an up-regulator of Nrf2 and exposure to LY294002, a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, suppressed the fucoxanthin-induced activation of Akt, Nrf2, resulting in decreased GCLC and GSS expression. In accordance with the effects on GCLC and GSS expression, fucoxanthin induced the level of GSH. In addition, fucoxanthin treatment recovered the level of GSH reduced by ultraviolet B irradiation. Taken together, these findings suggest that fucoxanthin treatment augments cellular antioxidant defense by inducing Nrf2-driven expression of enzymes involved in GSH synthesis via PI3K/Akt signaling.
fucoxanthin; NF-E2-related factor 2; oxidative stress; cytoprotection; PI3K/Akt; GCLC; GSS; GSH
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Competition of nuclear factor-erythroid 2 factors related transcription factor isoforms, Nrf1 and Nrf2, in antioxidant enzyme induction☆
Chepelev, Nikolai L.
Zhang, Hongqiao
Liu, Honglei
McBride, Skye
Seal, Andrew J.
Morgan, Todd E.
Finch, Caleb E.
Willmore, William G.
Davies, Kelvin J.A.
Forman, Henry Jay
Redox Biology
Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.
► Nrf1 knockout increased GCLC and GCLM expression in human bronchial epithelial cells. ► Overexpressed Nrf1 forms p120 or p65 increased GCLC- and GLCM-EpRE luciferase activity. ► Overexpressed Nrf2 competes with Nrf1 forms. ► Nrf1 forms are unchanged in the lungs of mice by age. ► Nrf1 forms are unchanged in exposure to nanoparticulate air pollution.
EpRE, electrophile response element; ER, endoplasmic reticulum; GCL, glutamate cysteine ligase; GCLC, catalytic subunit of GCL; GCLM, modifier subunit of GCL; HBE1, human bronchial epithelial cells; HO-1, heme oxygenase; Nrf1, nuclear factor-erythroid 2 p45 subunit-related factor 1; Nrf2, nuclear factor-erythroid 2 p45 subunit-related factor 2; nPM, nanoparticulate air pollution; Nrf1; Nrf2; Glutamate cysteine ligase; Electrophile response element; Air pollution; Phase II genes
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Increased Glutathione Synthesis Following Nrf2 Activation by Vanadyl Sulfate in Human Chang Liver Cells
Kim, Areum Daseul
Zhang, Rui
Kang, Kyoung Ah
You, Ho Jin
Hyun, Jin Won
International Journal of Molecular Sciences
Jeju ground water, containing vanadium compounds, was shown to increase glutathione (GSH) levels as determined by a colorimetric assay and confocal microscopy. To investigate whether the effects of Jeju ground water on GSH were specifically mediated by vanadium compounds, human Chang liver cells were incubated for 10 passages in media containing deionized distilled water (DDW), Jeju ground water (S1 and S3), and vanadyl sulfate (VOSO4). Vanadyl sulfate scavenged superoxide anion, hydroxyl radical and intracellular reactive oxygen species. Vanadyl sulfate effectively increased cellular GSH level and up-regulated mRNA and protein expression of a catalytic subunit of glutamate cysteine ligase (GCLC), which is involved in GSH synthesis. The induction of GCLC expression by vanadyl sulfate was found to be mediated by transcription factor erythroid transcription factor NF-E2 (Nrf2), which critically regulates GCLC by binding to the antioxidant response elements (AREs). Vanadyl sulfate treatment increased the nuclear translocation of Nrf2 and the accumulation of phosphorylated Nrf2. Extracellular regulated kinase (ERK) contributed to ARE-driven GCLC expression via Nrf2 activation. Vanadyl sulfate induced the expression of the active phospho form of ERK. Taken together, these results suggest that the increase in GSH level by Jeju ground water is, at least in part, due to the effects of vanadyl sulfate via the Nrf2-mediated induction of GCLC.
Jeju ground water; vanadyl sulfate; glutamate cysteine ligase; human Chang liver cells; erythroid transcription factor NF-E2
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Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction
Sacco, James C.
Trepanier, Lauren A.
Pharmacogenetics and genomics
NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA.
Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semi-quantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced.
Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06–1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (p < 0.0001, r = 0.42; p = 0.01, r = 0.23, respectively), and expression of both proteins correlated with one another (p < 0.0001; r = 0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency.
These studies indicate that while novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low frequency cSNPs only appear to minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.
sulfamethoxazole; sulfonamide hypersensitivity; cytochrome b5; NADH cytochromeb5 reductase; single nucleotide polymorphism
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Maternal Obesity and Tobacco Use Modify the Impact of Genetic Variants on the Occurrence of Conotruncal Heart Defects
Tang, Xinyu
Nick, Todd G.
Cleves, Mario A.
Erickson, Stephen W.
Li, Ming
Li, Jingyun
MacLeod, Stewart L.
Hobbs, Charlotte A.
PLoS ONE
Conotruncal heart defects (CTDs) are among the most severe birth defects worldwide. Studies of CTDs indicate both lifestyle behaviors and genetic variation contribute to the risk of CTDs. Based on a hybrid design using data from 616 case-parental and 1645 control-parental triads recruited for the National Birth Defects Prevention Study between 1997 and 2008, we investigated whether the occurrence of CTDs is associated with interactions between 921 maternal and/or fetal single nucleotide polymorphisms (SNPs) and maternal obesity and tobacco use. The maternal genotypes of the variants in the glutamate-cysteine ligase, catalytic subunit (GCLC) gene and the fetal genotypes of the variants in the glutathione S-transferase alpha 3 (GSTA3) gene were associated with an elevated risk of CTDs among obese mothers. The risk of delivering infants with CTDs among obese mothers carrying AC genotype for a variant in the GCLC gene (rs6458939) was 2.00 times the risk among those carrying CC genotype (95% confidence interval: 1.41, 2.38). The maternal genotypes of several variants in the glutathione-S-transferase (GST) family of genes and the fetal genotypes of the variants in the GCLC gene interacted with tobacco exposures to increase the risk of CTDs. Our study suggests that the genetic basis underlying susceptibility of the developing heart to the adverse effects of maternal obesity and tobacco use involve both maternal and embryonic genetic variants. These results may provide insights into the underlying pathophysiology of CTDs, and ultimately lead to novel prevention strategies.
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Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase
Franklin, Christopher C.
Backos, Donald S.
Mohar, Isaac
White, Collin C.
Forman, Henry J.
Kavanagh, Terrance J.
Molecular aspects of medicine
Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as γ-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique γ-carboxyl linkage from glutamate to cysteine and requires ATP and Mg++ as cofactors in this reaction. GCLM increases the Vmax and Kcat of GCLC, decreases the Km for glutamate and ATP, and increases the Ki for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.
Glutamate cysteine ligase; GCL; GCLC; GCLM; Glutathione; GSH; Post-translational
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Induction of Lung GSH and Glutamate Cysteine Ligase by 1,4-phenylenebis(methylene)selenocyanate and its Glutathione Conjugate: Role of Nuclear factor-erythroid 2-Related Factor 2
Emmert, Sans W.
El-Bayoumy, Karam
Das, Arunangshu
Sun, Yuan-Wan
Amin, Shantu
Desai, Dhimant
Aliaga, Cesar
Richie, John P.
Free radical biology & medicine
The synthetic organoselenium agent 1,4- phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG), are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamate cysteine ligase (GCL), through activation of antioxidant response elements (ARE) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular related kinase (ERK) (220%) and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), while the naturally-occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE reporter cells up to 3-fold for p-XSC and ≥5-fold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2 (siNrf2). The dependence of GCL induction on Nrf2 was confirmed in Nrf2 deficient mouse embryonic fibroblasts (MEF) where p-XSeSG induced GCL subunits in wildtype, but not Nrf2 deficient cells (p<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo, and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious inducer of GSH.
Organoselenium; p-XSC; p-XSeSG; glutathione; γ-GCL; Nrf2; MEF; antioxidant response element (ARE); HepG2-ARE; Lung cancer; Fisher 344 rat
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AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio)
Van Tiem, Lindsey A.
Di Giulio, Richard T.
Toxicology and applied pharmacology
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF + FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF + FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF + FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1 and GCLc after BkF + FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction.
zebrafish; polycyclic aromatic hydrocarbons (PAHs); oxidative stress; morpholino; aryl hydrocarbon receptor (AHR)
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Epigallocatechin-3-gallate Inhibits Growth of Activated Hepatic Stellate Cells by Enhancing the Capacity of Glutathione Synthesis
Fu, Yumei
Zheng, Shizhong
Lu, Shelly C.
Chen, Anping
Molecular pharmacology
Activation of hepatic stellate cells (HSC), the key effectors in hepatic fibrogenesis, is characterized by enhanced cell proliferation and overproduction of extracellular matrix. Oxidative stress promotes HSC activation. Glutathione (GSH) is the most important intracellular antioxidant, whose synthesis is mainly regulated by glutamate-cysteine ligase (GCL). We reported previously that (−)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. We hypothesize that this inhibitory effect of EGCG might mainly result from its antioxidant capability by increasing de novo synthesis of GSH. In this report, we observe that EGCG enhances the levels of cytoplasmic and mitochondrial GSH and increases GCL activity by inducing gene expression of the catalytic subunit GCLc, leading to de novo synthesis of GSH. Real-time polymerase chain reaction and Western blotting analyses show that de novo synthesis of GSH is required for EGCG to regulate the expression of genes relevant to apoptosis and to cell proliferation. Additional experiments demonstrate that exogenous transforming growth factor (TGF)-β1 suppresses GCLc gene expression and reduces the level of GSH in cultured HSC. Transient transfection assays and Western blotting analyses further display that EGCG interrupts TGF-β signaling by reducing gene expression of TGF-β receptors and Smad4, leading to increased expression of GCLc. These results support our hypothesis and collectively demonstrate that EGCG increases the level of cellular GSH in HSC by stimulating gene expression of GCLc, leading to the inhibition of cell proliferation of activated HSC in vitro.
Related Articles
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text:
If You're Thinking of Living in: Sloatsburg
By JERRY CHESLOW
Published: November 12, 1989
WHEN George Washington stopped at Sloatsburg, an old Rockland County joke has it, he told the residents not to change a thing - and they haven't.
But now a development boom has come to this village in the southwestern corner of Rockland County. Applications have been filed for permits for 365 new homes, which would increase the number of dwellings by 36 percent.
The new homes are expected to sell for between $150,000 for a two-bedroom condominium town house and $400,000 for a 3,000-square-foot house on three-quarters of an acre.
A 20-store shopping center is also being planned and construction is about to start on several plants in the light-industrial corridor along Sterling Mine Road.
''In 10 years, you'll hardly recognize Sloatsburg,'' said Mayor Samuel J. Abate. ''We're one of the last undeveloped areas of Rockland County and we've learned from everyone else's mistakes. So we'll make sure we have balanced development that also preserves the natural beauty of the village.''
The three-square-mile village is shaped like a circle with a flattened western side and is bisected by the Ramapo River, which flows through a deep gorge. Much of the land leading to the river is steep and rocky, which gives many houses steep lots and spectacular water views. The terrain may also have played a role in keeping two-thirds of the village in a natural state.
Mayor Abate said previous administrations stunted Sloatsburg's growth. ''They wanted to preserve the rural character of the village at all costs,'' he said. ''But staying small has its price. And that's why we have about the highest tax rate in the county. The only way to bring it down is to expand the tax base through development.''
According to Agnes N. Valentine, a broker with Melvin R. Smith Realtors in Sloatsburg, single-family homes are readily available. There are also about 175 rental apartments in two-family houses. Although there are no condominiums, permits have been sought for more than 100 such units.
Most houses are on lots of a third to half an acre, although some on Overlook Drive and Eagle Valley Road have much more land and sell for up to $400,000.
The building-permit review process often takes several years. After passing local review, any construction proposed within 150 feet of the Ramapo River must also be approved by the state's Department of Environmental Conservation.
Besides examining the possible environmental impact of each project, the village also examines hidden costs and requires developers to pay for all sewer and road improvements that relate to their properties, as well as $1,500 per new housing unit for the development of recreational facilities.
The only village recreational facility is a municipal ballfield leased from a land-development company, but the lakes, picnic grounds and hiking trails of Palisades Interstate Park are close at hand. The village has applied for $200,000 in state Environmental Quality Bond Act funds to acquire the ballfield and to open hiking trails, jogging paths and a boat launch along the Ramapo River.
BESIDES high taxes, residents complain most about the traffic along Route 17, Sloatsburg's main street, which is used by commuters from neighboring Orange County on their way to and from jobs in New Jersey or New York City. According to Mr. Abate, by 1994 a Gov. Thomas E. Dewey Thruway exit will be built in neighboring Tuxedo to ease congestion.
Sloatsburg has only 65 commercial establishments, including light industry, offices and about 25 stores concentrated on Route 17, among them farm markets and a few hunting and fishing equipment and several auto parts stores.
''The sense of community is why we moved here 13 years ago,'' said Jeanne M. McLoughlin. ''The old-timers are sometimes a bit distant. But if you want to get involved, and especially if you have kids, you get accepted pretty quickly.''
Mrs. McLoughlin said that she was also drawn to the Ramapo Central School District. It serves Sloatsburg as well as Hillburn, Montebello, Suffern, Wesley Hills and sections of Airmont, Ramapo, Tallman and Viola. The district has 3,637 students. Sloatsburg's pupils attend the local elementary school, which has a class size of less than 20, then go on to Suffern Junior High School and Suffern High School, where class sizes are in the low 20's.
According to Assistant Superintendent of Schools, Ticknor B. Litchfield, 75 percent of the students who attend Suffern High go on to college.
He said the district consistently scores in the 90th percentile on state tests, including the Pupil Evaluation Program for elementary school, the Preliminary Competency Tests for junior high school and the Regents Examinations for high school students.
''When people look for housing in the area, they look for the Ramapo Central School District,'' Mr. Litchfield said. ''We have a very good name. It's partly because we offer a very broad curriculum, which allows each student to tailor his program to his needs.''
The district has a wide-ranging computer literacy program, starting with kindergarten, where students are taught the graphics-based Logo computer language. There are junior high and high school courses in advanced computing, including word processing, computer-aided design and business computing. The district also offers a wide variety of courses for both the gifted and the handicapped.


text:
AUBURN, Ala. (AP) -- Auburn's players are describing the next two
games as "make or break" and "all or nothing."
That certainly could be true in the national championship
picture, but the No. 2 Tigers also face their two biggest rivals,
Georgia and No. 11 Alabama.
Auburn needs only to beat one of them to play in the
Southeastern Conference title game. Win both, and they most likely
head to Atlanta with a BCS championship shot on the line.
Auburn receiver Terrell Zachery says he feels like the next two
games mean "everything."
The Bulldogs must beat one of their two traditional rivals,
Auburn or Georgia Tech, to become bowl eligible. Bulldogs coach
Mark Richt says his players were probably turning thoughts to
Auburn even before Saturday's rout of Idaho State ended.
(Copyright 2010 by The Associated Press. All Rights Reserved.)


text:
- Report: #156651
Report - Rebuttal - Arbitrate
Complaint Review: Blue Haven Pools
Blue Haven Pools293 Goolsby Bl. / 2845 N. Military Trail #4 Deerfield Beach, Fort Lauderdale, West Palm Beach, Florida U.S.A.
Blue Haven Pools RIPOFF No Customer Service, Long Delays, Poor Workmanship, No Attention to Detail, Unending Excuses, Unfinished Results, Incompetent Management, No National Customer Care Deerfield Beach, Fort Lauderdale, West Palm Beach Florida
*Author of original report: Warranty not honored!
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It's been 16 month's since the day of signing and Blue Haven has yet to file accurate plans with the City Building department even though the pool does have water and is usable but totally unacceptable when compared to the high standards presented during the sales ride. At the moment the plans are supposedly still being redrawn and corrected by the architect yet no one in the organization is capable of communicating a date they will be finished and filed, which by the way is the norm for every step of the process.
I've also been advised that the local Blue Haven office has a reputation of often not completing the Inspection Finals which leaves me quite concerned.
During the building process I learned that the organization really has little to no organization and that if you expect construction to move along you must get heavily involved and consistently attempt to get Blue Haven's attention by contacting all possible levels of management starting with the salesman, the regional manger, and finally the National Customer Care line (which at first turned out to be an 800 number routed to the local office, until I was able to ferret out the real one located in San Diego and now Dallas, not that either had much effect).
To this day even after speaking with the National office several times all that has been offered is lip service and no known action to correct anything or to address a serious issue regarding additional charges falsely incurred.
All the work performed is by sub-contractors with little to no Blue Haven supervision or follow-up. It appears that someone at the local office has a list of steps and then schedules the work assuming the subs will show up and perform the work correctly which often is not the case.
For example, the steel was moved 3 times before being positioned correctly. It was actually even completely removed once and rebuilt from ground up. Plumbing also was missed and I had to contact the office repeatedly before anyone would come out and look and then after that the missing line was quickly and sloppily installed. The electrician left an outdoor plug non-functional and took several months before being able to get the pool light working and the work was not done as agreed. I have numerous other comments but let me wrap up the workmanship issues by just noting that each step on the checklist ended with issues of which some are yet to be resolved.
Beyond the shoddy workmanship during the construction let me also mention the damages, debris, and lack of respect to your personal property you can expect during the process. Anything on your property is subject to damage, be prepared to have gunite sprayed on anything (house, trees, bushes, etc) within several yards of the pool, the street will be damaged with cuts and gashes in the asphalt and also will be badly stained from leaking oil from the equipment and chemicals spilled, trash (drink containers, food wrappers, cigarette butts, etc..) and construction materials will be left scattered all around and do not completely get removed during the final pickup
In conclusion, if I ever have to contract a pool builder or recommend one in the future you can rest assured it will NOT be Blue Haven Pools especially the incompetent, unprofessional, disrespectful, discourteous, deceitful, and unresponsive staff in the offices located in South Florida.
Coral Springs, Florida
Click here to read other Rip Off Reports on Blue Haven Pools
This report was posted on Ripoff Report on 09/10/2005 07:21 PM and is a permanent record located here: http://www.ripoffreport.com/r/Blue-Haven-Pools/Deerfield-Beach-Fort-Lauderdale-West-Palm-Beach-Florida-33442-33409/Blue-Haven-Pools-RIPOFF-No-Customer-Service-Long-Delays-Poor-Workmanship-No-Attention-t-156651. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
Ripoff Report has an exclusive license to this report. It may not be copied without the written permission of Ripoff Report.
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#1 Author of original report
Warranty not honored!
AUTHOR: David - (U.S.A.)
SUBMITTED: Wednesday, June 09, 2010POSTED: Wednesday, June 09, 2010
As expected, the pool now has a defect which the warranty clearly indicated would be covered, but I'm told by Blue Haven that it is not covered.
Because the rip off franchise in South Florida was shut down, any and all written warranty guarantees they made are null and void! Corporate Blue Haven will not honor them even though they are the ones that forced the office to close.
All I can say folks, is do NOT, under no circumstances ever consider using anyone or anything with the name Blue Haven on it. That's all it is, a name only.
Coral Springs, Florida
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