I'm pressed for time to write this post, but if I don't get to it soon I'm afraid I won't at all, and that would be a shame because 3:10 to Yuma is a fantastic film. Definitely one I want to recommend. The first thing I did after seeing it was tell my stepfather to go see it. He loves "guy" movies and he loves great movies, and 3:10 to Yuma is definitely both those things.
The film is based on Elmore Leonard's short story and is a remake of the 1957 film starring Glenn Ford and Van Heflin. James Mangold directed the latest version. It's an excellent thing when you marry a genre like action or western with a filmmaker known for sensitive, complicated films that feature wonderful characters (Walk the Line, Cop Land, Girl Interrupted). You wind up with a unique take on genres that characteristically trade depth in favour of bullets and explosions.
3:10 to Yuma illustrates the point that all films, regardless of genre, benefit from strong character development. It's what makes the Bourne trilogy superior to the Die Hard series. And it's why Daniel Craig in Casino Royale was the most successful Bond to date.
The story follows the relationship between Ben Wade (Russell Crowe) and Dan Evans (Christian Bale). Okay, I realize it's not sounding like much of a "guy" movie yet. But hold your horses! Wade is a deadly outlaw. And Evans is part of a posse that's escorting Wade to Contention where he'll catch the 3:10 train to the Yuma prison. It's a dangerous job for Evans; more than one member of the posse meets with Wade's violent ways en route to Contention.
Seeing this movie, I felt like I was back in film school watching a William Wyler western. It's a classic western with the benefit of modern technology and all that it entails: excellent effects, sharp visuals, etc. But the reason it works so well is that the characters are believable. Crowe and Bale are a great pairing. Of the two, I'm partial to Crowe. Bale always strikes me as a little bit one note, although he was excellent in Batman Begins. But both give top-notch performances.
I could go on. But not tonight. See this film!


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Anthony V. Guarisco
Anthony Vito Guarisco, 81, passed away peacefully on Saturday, August 31.
Born July 18, 1938 in Chicago, IL, the youngest child and only son of John and Anna (Tramonte) Guarisco, his three older sisters were additional mother figures to him. He grew up in the city of Chicago near Humboldt Park. Upon leaving high school, he became an apprentice plumber, working with his brother-in-law Joe Donato.
While visiting a family-owned resort on Newton Lake, he met the love of his life, Eleanor (Baker). On November 30, 1957, the two were wed at St. Sylvester Church in Chicago. Tony was a plumber in Chicago until buying a tavern in rural Crivitz (located 13 miles West of Crivitz on Cty. W.) and called it the Top Wop. They owned Tony and Ellie's Top Wop from 1978 until 1983.
Tony and Ellie raised their 5 children in Illinois and Wisconsin. They left the tavern in 1983 and returned to the Chicago area, where Tony worked with his son, Joe, running Guarisco Plumbing until retiring and moving back to Crivitz in 2001.
More than anything, Tony loved his family. Everyone in the family knew if they needed something he would do anything for them. If you were family, or considered family, you didn't have to ask twice or explain what had happened, he was there doing whatever needed to be done.
His 15 grandkids loved to go fishing with grandpa at "the pond" and listening to the many colorful stories he had to tell. He would often break into song whenever the mood struck him and he would sing his own version of "Froggy Went a Courtin" and "Okeechobee". If it was your birthday, friends and family would get an extra treat--a phone call from Tony, who would sing "Happy Birthday" off key.
Other than his family, his garden was his pride and joy. He spent countless hours planting, watering, and harvesting his crops. He enlisted the help of many for those jobs, but loved the company and sharing what he grew. Many friends and family enjoyed a melon from his garden while sitting in his "office" with him.
Tony was an Endowment Lifetime Member of the NRA and taught all of his grandkids to hunt. Family knew they could always count on an open deer stand in the Sicilian Swamp. He had a smile and a story for everyone he met.
Tony is survived by his wife of 61 years, Eleanor; children, John (Debbie) Guarisco of Middle Inlet, Cathy (Roy) Woltmann of Norridge, IL, Vicki Guarisco of Crivitz, Joe (Carrie) Guarisco of Crivitz and Peggy (Jim) Zielinski of Crivitz, along with 15 grandchildren, Gina (Tom), Nikki (Brett), Tommy (Hannah), Anna (Drew), Tony, Brian (Cherish), Gary, Joey (Breanne), Anthony (Ashlei), Alyssa (Erik), Francesca (James), Becky (Brian), Kelly (Matt), Sarah and Alex (Amber); 23 great-grandchildren; his brother-in-law, Jim (Cheryl) and sister-in-law, Nancy, along with many nieces and nephews.
He was preceded in death by his parents, John and Anna; his sisters, Mamie (Joe) Donato, Mary (Al) Binsfield and Genevieve (Bob) Batson; his father-in-law (Sidney) and mother-in-law (Catherine); his sister-in-law, Barb (Harold) and a nephew, Steven.
Visitation will be held Thursday, September 5th from noon until time of the service at 3PM at Rhodes Charapata Funeral Home, Crivitz, WI. With Graveside service to follow.

redpajama_set_name: RedPajamaCommonCrawl


text:
Hellas Verona 2022/23 adults' home match jersey
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The Hellas Verona 2022/23 home jersey offers a nod to the club's past. The idea came from a milestone anniversary: March 2023 marks the 120th anniversary of the club's foundation. Both the first match jersey worn in 1903, and the jerseys from the Veneto club's early years all featured a shield badge with a cross. The symbol has been reinstated and reproposed in this new shirt, where it is embossed on the front. VERONA is embroidered in yellow on the back of the collar. The piping at the sides and on the back of the shoulders adds a touch of refinement and lightness to the garment.
The jersey will be available for delivery as of the 13th of July but it is already possible to reserve it by placing a pre-order. If you order items which are already available and pre-order items, these will be sent together in one single delivery on the same date as the pre-order items. The total amount will be debited when the order is complete.
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I can spend hours in stores like Sephora or Forever 21 and end up not buying anything. I'm the kind of shopper who tries on so many things but in the end asks myself, "do I really need this?". Unless it's arm candies! I'm a sucker for arm candies!
I have a huge obsession with pink, practically all shades of pink! There was a period of time when I was ashamed to admit that I like the colour because it was too girly or feminine and I started to pretend to like the colour black but oh gosh, just, no.
I actually hate birds. They're loud and noisy and I have a weird phobia that birds might suddenly attack me by pecking on my head. They also have this goddamn evil look in their eyes, omg I kennut sia guys. I KENNUT.
I am a mega fan of One Direction although I don't fangirl whenever I their songs are on the radio because I find it damn annoying when other people go crazy when their favourite songs are on.
I'm used to really love cupcakes until I tried donuts from Krispy Kreme and afterwards I became obsessed with donuts, preferably the glazed ones!
My wardrobe consists of roughly five different coloured pants which are super comfy!
I have bought a total of three fish eye lens for my phone as I keep on losing them, hehe!


Jen Neal: I am a Brooklyn-based jewelry and fiber artist that makes woven wall hangings, tassel necklaces and teaches weaving workshops. I spend my days working to support the human rights movement and promoting documentary films that can challenge people to find ways to create change in their communities. Each night after work and during any spare moments I can find throughout the day - I put my heart to work designing and creating. I'm also a mother to two beautiful and hysterical little boys ages 1 and 3.
SS: How did you fall in love with Fiber?
JN: I've always been surrounded by great makers. My great grandfather was a tailor and I grew up with women around me who would take pride in showing me the things they made with a sewing machine or a pair of knitting needles. Being able to make things with your hands was always a romantic notion to me. It felt empowering as a little girl to be able to turn some embroidery thread into a friendship bracelet, to work with a beading loom, creating latch hook rugs and making my own clothes. It's always been a part of how I relate to the world. It also allowed me at an early age to show off my entrepreneurial spirit :) I would sell my friendship bracelets, chokers, and other handmade items in the lunchroom at school or in a stand set up in my suburban driveway. Just as it did back then as a little girl, making jewelry by hand brings me so much joy and confidence. It may sound dorky, but seeing my work worn proudly by other women is the best feeling in the world. It's a true honor to see my work making other women feel beautiful and powerful in some small way.
I went to art school and studied fashion, illustration, and painting before I graduated with a degree in video art. Since my day job focuses so much on the many injustices people across the globe are facing, being able to come home and reconnect with fibers is very therapeutic. I find the community of fiber artists in Brooklyn and beyond so incredibly inspiring. There are so many talented women out there weaving and making fiber jewelry including my friend and fellow jewelry/macrame artist Alisha Rodriguez. Most of us have kids and use the income we bring in to support our families, other women makers and giving back to charities we care about.
SS: What techniques do you use to create your jewelry? What is your process?
JN: Each tassel necklace I make is made for the most part from ethically sourced fibers. The cord for my necklaces comes from fashion industry off-cuts in Europe and the fibers in the tassels themselves, for the most part, come from recycled denim, cotton, and vintage fibers. For that reason, each piece is unique and once I run out of a certain color or fiber - it's gone for good. I crochet each necklace cord, then take the time to carefully select fibers and wrap the tassel by hand.
SS: Any specific inspiration behind your creations?
Maybe it's the inspiring ways in which women right now are coming together and standing up for their rights, photos of my mother raising me in the 1970s, or the inspiring female fiber artists of the 1960's/1970's I have been obsessing over - but I design my tassel necklaces with strong women in mind - while paying homage to the ladies of the 1960's-70's fighting the patriarchy with their high-waisted jeans. The tassel necklaces look great with a pair of faded jeans, a simple t-shirt or a shift dress.
SS: We are a New York brand and you are a New York/Brooklyn-based designer, tell us some of your favorite places to eat, shop and hang out.
JN: I have lived in Brooklyn for the last 16 years, and feel like I've grown up here. I moved here before I turned 21 and now find myself a lady with two kids and some gray hairs. My grandmother was born on Washington Ave in Prospect Heights, and both my grandfathers went to school in Clinton Hill and Fort Greene when they were teens.
A perfect Sunday for me would include grabbing a coffee and pastry with friends at Stonefruit Espresso and Kitchen (where I also host my weaving workshops!) They have a plant shop in the back that is the perfect place to send off some work emails while sipping tea and listening to great music. The restaurant Buttermilk Channel in Carroll Gardens has the best friend chicken and waffles and is my go-to spot for special birthday dinners. Paired with a glass of Rose and their honey/sea salt popovers theirs is pretty much my favorite meal on the planet. Across the street from Buttermilk Channel is Frankie's Spuntino, another favorite dinner spot. A few blocks from our house in Bed-Stuy is a spot that my husband and I love to take our family called Peaches Shrimp and Crab. When we go there our kids often stroll around like they own the place. On our corner, we also have the amazing Speedy Romeo (amazing wood fired pizza), and Clementine Bakery, (a vegan bakery and sandwich shop). On the rare late night when my husband and I go all out - we love dancing at Friends and Lovers and stopping by Chilo's for some margaritas and tacos.
Now that we have two kids under 4 years old, our hang out spots are somewhat limited to the spots where they can run free. We love the community gardens in our neighborhood, including Greene Acres - where they keep a few chickens that my kids love to play with and feed berries.
My favorite places to shop in Brooklyn include Meg (of course!), Purl Soho (for fibers and sewing notions), Sweet William (for kids clothes) and the Brooklyn Flea for vintage.Find Jen's necklaces in your local store.


text:
Advances in Biological Chemistry
Vol.4 No.1(2014), Article ID:42711,9 pages DOI:10.4236/abc.2014.41005
N-acetylcysteine amide protects against dexamethasone-induced cataract related changes in cultured rat lenses
Shakila Tobwala1, Eylem Y. Pinarci2, Yasaswi Maddirala1, Nuran Ercal1*
1Department of Chemistry, Missouri University of Science and Technology, Rolla, USA
2Department of Ophthalmology, Baskent University, Istanbul, Turkey
Email: *nercal@mst.edu
Copyright © 2014 Shakila Tobwala et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accordance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intellectual property Shakila Tobwala et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian.
Received 5 December 2013; revised 10 January 2014; accepted 21 January 2014
Dexamethasone; Cataracts; Oxidative Stress; Antioxidant; N-Acetylcysteine Amide
Glucocorticoids (GCs) are one of the most widely used immunosuppressive and anti-inflammatory agents. However, their long term and systemic use is associated with adverse drug reactions including posterior subcapsular cataracts as one of its ocular complications. Balanced redox state is crucial for maintenance of lens transparency, and a high content of glutathione (GSH) in the lens is believed to play a key role in doing so. Depletion of GSH is implicated in the etiopathogenesis of dexamethasone-induced cataracts and, therefore, the present study was sought to evaluate the efficacy of a novel thiol antioxidant, N-acetylcysteine amide (NACA), in preventing dexamethasone-induced cataractogenesis. Cataract formation was induced by incubation of rat lenses with 5 µM dexamethasone. To assess whether NACA had a significant impact on dexamethasone-induced cataracts, the rat lenses were divided into four groups: 1) control group (Dulbecco's Modified Eagle Medium (DMEM), 2) dexamethasone group (DMEM with 5 µM dexamethasone), 3) NACA-only group (50 µM NACA solution), and 4) NACA pretreatment group (50 µM NACA for 6 hours followed by 5 µM dexamethasone only for 18 hours). Lenses were cultured for 7 days at 37˚C under 5% CO2. Lenses were evaluated daily using a dissecting microscope and photographed and graded for the development of opacity. The rat lenses in both the control and the NACA-only groups were clear, whereas all lenses within the dexamethasone-only group developed well-defined cataracts. Overall observations indicated that NACA inhibits cataract formation by limiting lipid peroxidation and increasing the ratio of GSH/GSSG in lens. Therefore, NACA can be developed into a potential adjunctive therapeutic option for patients undergoing therapy with GCs to inhibit glucocorticoid-induced cataracts.
Glucocorticoids (GCs) are steroid hormones that play a role in physiological processes and are widely used as immunosuppressive and anti-inflammatory agents in the treatment of many clinical conditions, including rheumatoid arthritis, asthma, autoimmune diseases, and various ocular diseases [1,2]. However, their clinical use is restricted due to a wide range of complications associated with their long-term topical and systemic use. One of the ocular complications of glucocorticoid toxicity is the development of posterior subcapsular cataracts (PSCs) [3-8]. Unfortunately, certain patients cannot avoid longterm steroid therapy and therefore, development of adjunctive therapeutics for the prevention of steroid-induced cataracts is highly desirable.
Oxidative stress and depletion of GSH are implicated in the etiopathogenesis of glucocorticoid-induced cataracts [9-14]. Under physiological conditions, the lens utilizes its various antioxidant defenses to effectively protect itself and maintain the reduced state of thiols, which is essential for retaining clarity of the lens. However, under oxidative stress, depletion of GSH disturbs the thiolredox status. This sets off a cascade of oxidative damage resulting in the oxidation of all major macro-molecules in cells, including lipids, proteins, and nucleic acids, and eventually in the disruption of function and integrity of lens cells [15-19]. Oxidative damage to proteins may lead to structural and functional changes, including conformational changes resulting in inhibition of enzymatic and binding activities, fragmentation, denaturation, aggregation, altered gene expression and regulation, and modulation of cell signaling [20]. In addition, lipid hydroperoxides may cause changes in membrane permeability [21-24] along with uncoupling of the membranebound enzyme Na+/K+-ATPase and oxidative inhibition of Ca2+-ATPase [25,26], and DNA damage [27]. Changes in the redox ratio of GSH lead to crosslinking, aggregation, insolubility, and fragmentation of crystalline proteins, which results in the formation of cataracts [12, 28-31]. Epidemiological studies have documented loss of GSH in various types of cataracts [32-34]. A decrease in GSH levels after glucocorticoid exposure [35,36] along with an increase in the levels of lipid peroxide in the lens [36] has been reported. Furthermore, protection by antioxidants like vitamin E and ascorbic acid [7,37] against damage caused by a soluble corticosteroid suggests the role of oxidative stress in steroid-induced cataract formation.
At present, the treatment for cataracts requires surgery to remove the natural lens that has developed opacification, replacing it with a synthetic lens to provide transparency. Although cataract surgery is considered to be one of the safest procedures available, treatment is relatively expensive and there is a significant rate of postsurgical complications, including the development of a posterior capsular opacification [38]. Since depletion of GSH is hypothesized to be a key initiating event in the development of PSCs, the use of a GSH prodrug as an adjunctive therapeutic agent would be an effective, nonsurgical treatment to prevent and treat cataracts. However, progress in this area has been modest. Although studies have indicated that antioxidants like N-acetyl carnosine and N-acetyl cysteine (NAC) may ameliorate the risk for cataracts [39,40]. NAC is not highly bioavailable and does not readily penetrate into cells, thereby requiring fairly high doses that can increase the side effect profile. A potential candidate that possesses far better characteristics for development as an ophthalmologic agent to address oxidative stress damage is the low molecular weight thiol antioxidant, N-acetylcysteine amide (NACA). NACA's characteristics as a drug were improved over NAC by neutralizing the carboxylic group of NAC, which makes the NACA molecule more lipophilic and therefore enhances its ability to penetrate cellular membranes. The enhanced ability to penetrate cells allows NACA to be administered at a lower dose than NAC, giving the drug a greater therapeutic index and lowering the risk of side effects that traditionally have been associated with higher doses of NAC [41,42]. NACA is an excellent source of sulfhydryl (SH) groups that can be converted by the cells into metabolites capable of stimulating GSH synthesis. The molecule also can promote intracellular detoxification and act directly as a free radical scavenger. NACA acts as a carrier of NAC and its antioxidant and free radical scavenging abilities are equal to or better than those of NAC [43].
Promising results with NACA [44-51] in various oxidative stress-related disorders encouraged us to investigate the protective role of NACA in the prevention of dexamethasone-induced cataracts. Our data showed that NACA inhibits dexamethasone-induced cataract formation by limiting lipid peroxidation and increasing the ratio of GSH/GSSG in lenses. NACA can potentially be developed into a promising adjunctive therapeutic option for patients undergoing therapy with glucocorticoids.
2.1. Materials
Dexamethasone and N-(1-pyrenyl)-maleimide (NPM) were obtained from Sigma-Aldrich (St. Louis, MO). High performance liquid chromatography (HPLC) grade solvents were purchased from Fisher Scientific (Fair Lawn, NJ). NACA was gifted by Dr. Glenn Goldstein (David Pharmaceuticals, New York, NY). All other chemicals were bought from Sigma-Aldrich (St. Louis, MO).
2.2. Ex Vivo Rat Lens Culture and Drug Treatment
All animal procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and by the Animal Care and Use Protocol Review Committee at the Missouri University of Science and Technology. For rat lens culture, eyes from 21-dayold Sprague-Dawley male rats were enucleated to expel the lens using plastic-coated forceps and fine scissors. Eyes were immediately transferred to Dulbecco's MEM (pH 7.2; Sigma, St. Louis, MO), containing 0.1% bovine serum albumin (BSA; GibcoBRL, Grand Island, NY) and antibiotic solution (GibcoBRL; 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B) [52]. The culture media was changed daily. Approximately 24 hours after the preparation of organ cultures, clear lenses were selected and were randomly divided into four groups: 1) control group (DMEM), 2) dexamethasone group (DMEM with 5 µM dexamethasone), 3) NACA-only group (50 µM NACA in DMEM), and 4) NACA pretreatment group (pretreatment with 50 µM NACA for 6 hours followed by 5 µM dexamethasone only for 18 hours in DMEM). Lenses in each group were cultured in DMEM media as detailed above for 7 days at 37˚C under 5% CO2. Lenses were evaluated daily using a dissecting microscope and photographed to check for the development of opacity.
2.3. Intracellular Glutathione (GSH) Measurement
Intracellular GSH content was determined by reverse phase HPLC, according to the method developed in our laboratory [53]. Lens samples were homogenized in serine borate buffer (SBB). Twenty microliters of this homogenate were added to 230 µl of HPLC grade water and 750 µl of NPM (1 mM in acetonitrile). The resulting solutions were incubated at room temperature for 5 min. The reaction was stopped by adding 10 µl of 2 N HCl. The samples were then filtered through a 0.45 µm filter (Advantec MFS, Inc. Dulin, CA, USA) and injected onto the HPLC system. 5 µl of the sample were injected for analysis using a Thermo Finnigan TM Spectra SYSTEM SCM1000 Vacuum Membrane Degasser, FinniganTM SpectraSYSTEM P2000 Gradient Pump, FinniganTM SpectraSYSTEM AS3000 Autosampler, and FinniganTM SpectraSYSTEM FL3000 Fluorescence Detector (λex = 330 nm and λem = 376 nm). The HPLC column was a Reliasil ODS-1 C18 column (Column Engineering, Ontario, CA, USA). The mobile phase was 70% acetonitrile and 30% water and was adjusted to a pH of 2.5 through the addition of 1 ml/L of both acetic and o-phosphoric acids. The NPM derivatives were eluted from the column isocratically at a flow rate of 1 ml/min.
2.4. Total Glutathione and Glutathione Disulfide (GSSG) Measurement
Total glutathione content was determined by reverse phase HPLC. Lens samples were homogenized in SBB. Twenty microliters of this homogenate were added to 60 µl of NADPH (2 mg/ml) in nanopure water and 20 μl of 1 unit/ml glutathione reductase were added to reduce GSSG. After 10 min of incubation at room temperature, the treated samples were diluted with 150 μl H2O, and then immediately derivatized with 750 μl of 1.0 mM NPM. The samples were analyzed as detailed for the determination of GSH using reverse phase HPLC. Data from the original GSH levels and the total current GSH levels in each sample were subsequently used to calculate the levels of GSSG present in each sample [54].
2.5. Determination of Glutathione Reductase (GR) Activity
Glutathione reductase is the enzyme responsible for recycling GSSG into GSH via a reduction mechanism, utilizing both GSSG and NADPH as a substrate. The activity of this enzyme was determined using a commercial kit from OxisResearch (Portland, OR, USA). The oxidation of NADPH to NADP+ was accompanied by a decrease in absorbance at 340 nm, providing a spectrophotometric means for monitoring the enzyme activity of GR. The activity of GR in cells was determined by adding homogenate to a solution containing both GSSG and NADPH and then recording the absorbance as a function of time at 340 nm. The rate of decrease in the A340 was directly proportional to the GR activity in each sample.
2.6. Lipid Peroxidation Measurement
Malondialdehyde (MDA) is a thiobarbituric acid reactive substance (TBARS). The extent of lipid peroxidation was determined by measuring concentrations of TBAMDA complex. Lens homogenate (350 μl), 100 μl of 500 ppm butylated hydroxytoluene, and 550 μl of 10% trichloroacetic acid were combined, and the suspension was boiled for 30 min. An aliquot (500 μl) of the supernatant was removed and 500 μl of thiobarbituric acid added. From this solution, 500 μl were removed and added to 1.0 ml of n-butanol. This mixture was vortexed, and centrifuged for 5 min at 110 × g to facilitate phase separation. Fluorescence was then measured (λex = 515 nm and λem = 550 nm) [55].
All reported values were represented as the mean ± S.D. of quadruplets. Statistical analysis was performed using the GraphPad Prism software (GraphPad, San Diego, CA). Statistical significance was ascertained by one way analysis of variance, followed by Tukey's multiple comparison tests. Values of p < 0.05 were considered significant. In the figures, "*" represents a significant difference in comparison with the control group, and "#" represents a significant difference in comparison with the Dex-only group.
3.1. Prevention of Dexamethasone-Induced Cataracts by NACA
To examine the effects of dexamethasone on the lens, we used an ex vivo rat lens model. We isolated rat lenses and treated them in organ culture to cause steroid-induced cataracts directly. The lenses were incubated with 5 µM dexamethasone and morphologic changes in the whole lenses were recorded photographically with a dissection microscope. Opacity first appeared at day 3 in the lenses incubated with dexamethasone. All lenses in the dexamethasone group developed cataracts by day 7. In contrast, only 25% of the lenses developed cataracts in the NACA pretreatment group. Untreated control lenses remained transparent until day 7 (Figure 1).
3.2. Effect of NACA on Intracellular GSH Levels in Dexamethasone Treated Cultured Rat Lens
To support our hypothesis that GSH depletion induces cataract formation in the Dex-treated group, we measured the levels of intracellular GSH. Figure 2 shows the effect of Dex on lens GSH levels in the presence and absence of NACA. A 7-day exposure with 5 μM of Dex decreased the GSH level to 21% of that of the control. A pretreatment with 50 μM of NACA increased the GSH level close to control.
3.3. Effect of NACA on Oxidized Glutathione (GSSG) Levels and GSH/GSSG Ratio in Dexamethasone Treated Cultured Rat Lenses
GSSG levels in the lenses of the Dex-only group were found to have significantly increased by approximately three times the GSSG levels in the control group. The amounts of GSSG found in lenses with NACA pretreatment were significantly lower compared to the Dex-only group and were close to that of the control group. A graph with these results is shown in Figure 3. An interesting result was obtained by observing the ratio between the GSH and the GSSG levels in the lenses of each group. As expected, the control group was found to have the highest ratio of GSH to GSSG. The ratio dropped to about 6% of control in the Dex-only group (Figure 4). However, NACA pretreatment increased this ratio significantly to approximately 50% of the control group value.
Figure 1. Appearance of cultured rat lens with dexamethasone and NACA at day 7. Opacity was observed only in the Dex group at day 7 (Figure 1(c)), which was significantly prevented by pretreatment with NACA. (a) Control; (b) NACAonly; (c) 5 µM Dex; (d) 5 µM Dex + 50 µM NACA. All reported values were represented as the mean ± S.D. (n = 8).
Figure 2. Intracellular GSH levels in lenses after treatment with dexamethasone and NACA. GSH levels were measured after 7 days of treatment for control, NACA, Dex, and Dex + NACA groups. Exposure to Dex (5 μM) significantly decreased intracellular GSH level. Pretreatment with NACA (50 μM), prevented such a dramatic decrease. The NACA-only treated group showed no significant difference when compared to the control. *p ≤ 0.05 compared to the control group. All reported values were represented as the mean ± S.D. of quadruplets.
Figure 3. Intracellular GSSG levels in lenses after treatment with dexamethasone and NACA. The GSSG level was significantly higher in the Dex-only group than in the control. This GSSG level was significantly reduced upon pretreatment with NACA. The NACA-only treated group showed no significant difference when compared to the control. *p ≤ 0.05 compared to the control group, and #p ≤ 0.05 compared to the Dex group. All reported values were represented as the mean ± S.D. of quadruplets.
3.4. Effect of NACA on Glutathione Reductase (GR) Activity in Dexamethasone Treated Cultured Rat Lenses
GR is a key antioxidant enzyme involved in maintenance of cellular GSH homeostasis. It reduces GSSG back to the reduced form, GSH. A significant reduction in the activity of GR was observed upon Dex treatment. However, NACA pretreatment increased the activity of GR (Figure 5).
3.5. Effect of NACA on Lipid Peroxidation Byproduct: MDA
MDA was used as an index of lipid peroxidation (LPO). Dex-treated lens had significantly higher levels of MDA, as compared to those of the control (Figure 6). Pretreatment with 50 µM of NACA completely reduced this increase, with MDA levels becoming nearly the same as those of the control, and with a p value of <0.05, as compared to that of the control. The NACA-only treated group showed no significant difference when compared to the control.
Posterior sub-capsular cataract is one of the ocular complications of glucocorticoid toxicity. Despite a well-established link between the use of GC's and cataracts, treatment with glucocorticoids cannot be avoided in some cases. GC-induced cataract formation is directly attributed to oxidative stress that occurs within the lens. Oxidation, which can be caused by an overabundance of oxidative stress generators, such as molecular oxygen, hydrogen peroxide, and free radicals, produces a major insult upon the lens, which can lead to the loss of GSH, LPO, and a decrease in antioxidant enzyme activity
Figure 4. GSH/GSSG ratio in lenses after treatment with dexamethasone and NACA. The GSH/ GSSG ratio in the Dex only group was significantly lower than in the control group. This GSH/ GSSG ratio was significantly increased upon pretreatment with 50 μM NACA. The NACA-only treated group showed no significant difference when compared to the control. *p ≤ 0.05 compared to the control group, and #p ≤ 0.05 compared to the Dex group. All reported values were represented as the mean ± S.D. of quadruplets.
Figure 5. Glutathione reductase activity in lenses after treatment with dexamethasone and NACA. GR activity was significantly lower in the Dexonly group than in the control group, while NACA pretreatment increased its activity. The NACAonly treated group showed no significant difference when compared to the control. *p ≤ 0.05 compared to the control group, and #p ≤ 0.05 compared to the Dex group. All reported values were represented as the mean ± S.D. of quadruplets.
Figure 6. MDA levels in rat lenses after treatment with Dex and NACA. It was found that, after 7 days of Dex treatment, the MDA levels significantly increased. Dex (5 μM) induced a significant increase in the MDA level. Pretreatment with 50 µM of NACA decreased lipid peroxidation significantly. The NACAonly treated group showed no significant difference when compared to the control. *p ≤ 0.05 compared to the control group, and #p ≤ 0.05 compared to the Dex group. All reported values were represented as the mean ± S.D. of quadruplets.
[56-58]. GSH is an indispensable and primary lenticular antioxidant [12]. A wide body of evidence indicates loss of GSH because of its oxidation to GSSG, since its levels increase drastically once cataracts develop. Therefore, an alternative method for treating or preventing the occurrence of GC-induced cataracts would be through the use of a GSH prodrug. With this background, we evaluated the effects of a novel antioxidant and a potent GSH prodrug, N-acetylcysteine amide (NACA), in the prevention of cataracts induced by Dex in rat lenses. Results from morphological observations indicated that NACA was able to reduce the opacification of the lens within this exvivo dexamethasone-induced cataract model (Figure 1).
As discussed earlier, GSH is an essential lenticular antioxidant and is present in high concentrations in the lens, providing a first line of defense against oxidative damage [59], as well as playing an important role in antioxidant defense and redox regulation [60]. Results from this study indicate that treatment with dexamethasone decreased lenticular GSH and the GSH/GSSG ratio significantly. In addition, it increased GSSG levels significantly. However, pretreatment with NACA significantly increased the ratio of GSH/GSSG by decreasing the levels of GSSG. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a representative marker of the antioxidative capacity of a cell. Depletion of GSH could be due to several possible mechanisms, including but not limited to the efflux of GSH from the lens, sequestration of GSH by incorporation into mixed disulfide aggregates, enhanced consumption of GSH in the process of detoxification of ROS, and downregulation of enzymes involved in GSH biosynthesis. Another possible explanation for the decrease in GSH levels under oxidative stress is the reduced GR activity. Some studies have indicated that loss of GSH will directly affect the activity of the GSH-dependent enzyme GR. This enzyme plays an important role in GSH homeostasis. It has been reported that, under oxidative stress, the protein sulfhydryl (protein-SH) groups are lost [61], which are essential for enzyme activity [62]. Under such circumstances, GSH is not regenerated, so depletion of GSH indicates that the tissue is undergoing oxidative stress. The decreased activity of GR seen in our experiments with Dex treatment supports our hypothesis of GSH depletion upon treatment with Dex. A decrease in the lenticular GSH levels has been observed upon treatment with glucocorticoids [13,35,63]. In vivo treatment of rabbits with the Dex eye drops, as used for cataract surgery, reduced GSH levels in the lens [63]. In addition, free radical scavengers have also been reported to prevent GC-induced cataract formation [64-66] by increasing GSH levels and decreasing LPO. Decreasing the levels of GSH, when reactive oxygen species are present, can trigger a cascade of further oxidative damage, such as LPO, which has been associated with the formation of cataracts in patients [67-70].
The extent of LPO was determined in this study by measuring the amount of MDA, a by-product of LPO, within the lens. Unavailability of GSH as a substrate for glutathione peroxidase stalled the process of lipid peroxide decomposition and, thus, increased the levels of MDA in the Dex-treated group. NACA supplied an adequate amount of GSH as a substrate for glutathione peroxidase to effectively decompose lipid peroxides in the rats, reducing MDA levels (Figure 6). The multiple roles of NACA in preventing cataract formation include direct scavenging of free radicals, providing cysteine for GSH synthesis, and nonenzymatic reduction of the preexisting toxic GSSG into GSH.
Our results suggest that NACA can prevent the formation of dexamethasone-induced cataracts by directly and indirectly maintaining the GSH/GSSG ratio in healthy lenses, allowing the lens to better cope with oxidative stress. NACA could confer a protective effect by providing a substrate for the generation of GSH, maintaining antioxidant levels within the lens and, possibly, through disulfide-exchange mechanisms. Treatment with NACA may prove to have a major therapeutic role in dexamethasone-induced cataracts. In future studies, we will focus on the prophylactic role of NACA in GC-induced cataract formation and investigate the development of a topical formulation for the application of this antioxidant in an in vivo model.
Dr. Ercal is supported by 1R15EY022218-01A1 from the National Eye Institute (NEI), National Institutes of Health (NIH). The contents of this paper are solely the responsibility of the authors and do not represent official views of the NIH. The authors appreciate the efforts of Barbara Harris in editing the manuscript.
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text:
Sparky's owner is Ryan Maher, a young entrepreneur and South Dakota state senator.
South Dakota's Best Breakfasts
By Katie Hunhoff
"A timid salesman has skinny kids," quipped a sales consultant at a recent business meeting in Sioux Falls.
That may be especially true for salesmen in sparsely populated South Dakota, where you can literally run out of prospects and even restaurants. So the smart traveling salesman of the prairie makes the most of every day, every town, every mile.
And the experienced salesman knows the advantages of starting the day right, with a tasty breakfast enjoyed in a place where the locals meet — so we asked a few road veterans to share their favorite breakfast establishment as a travel tip for the rest of us.
Joie's Cafe — Winner
Although Wayne Hopkins of Brookings sells electrical and air conditioning parts for Nielsen's in a four state area, he chose a restaurant in his home town of Winner. "In the winter I'd go in the cafe, just a block from my school, to have a hot chocolate and warm up. It still looks the same as I remember it 30 years ago," Hopkins says. His favorite item is the breakfast burrito.
Brock Green succeeded his father-in-law at Joie's years ago. Special recipes for biscuits and gravy and made-from-scratch pancakes haven't changed. He even has his own specialty sausage, made just for Joie's at the local Super Duper Store.
The 140-seat Main Street cafe is a Winner mainstay that was called Sargent's when Hopkins was growing up. Visitors are welcome to sit at the businessman's roundtable, where locals shoot dice to see who picks up the noon tab. But be careful. "Usually it's the new guy or the guy who only had soup that gets nailed," laughs Green. Call 605-842-3788.
ALASKA CAFE — Lemmon
Lemmon is South Dakota's northernmost city, but it's still a far cry from the tundra so travelers are surprised to see the Alaska Cafe sign on Highway 12 and they often stop to pose for pictures.
Inside, they get an even better taste of the Land of the Midnight Sun. Pictures of grizzly bears, moose, the Bering Strait and North Pacific fishing boats grace the walls, and proprietor Laura Casey — who runs the cafe with her daughter, Breanna Thomas — has a big compass, the only surviving artifact of her father's commercial halibut boat that was lost in a storm. Several years after the accident, Laura's parents moved to Lemmon and she followed seven years ago and opened the restaurant.
Amy Pravecek of Winner chose the Alaska Cafe in Lemmon as her favorite breakfast spot because "everything on their menu is wonderful and the cafe is full of friendly locals who are always willing to visit," she says.
Pravecek is the territory manager for Phizer in western South Dakota. She travels West River back roads visiting veterinarians, animal health distributors, farmers and ranchers, telling them about Phizer's vaccination programs.
Alaska Cafe serves breakfast from 7 a.m. to 2 p.m. The big breakfast is a country fried skillet with scrambled eggs, hash browns, chicken fried steak, country gravy and cheddar sauce. Pancakes are the size of big plates.
Pravecek likes to dine on the biscuits and gravy and then take a little walk through the petrified wood park across the street. She also recommends visiting Lemmon Livestock sale barn if you are in town on a Wednesday. Call 605-374-7588.
SPARKY'S — ISABEL
Sparky's operates from a nondescript building on Isabel's Main Street, which is busier than you might expect because it also sits along S.D. Highway 65, a north-south corridor that cuts across West River country.
Operated by Ryan Maher, a young entrepreneur and Republican state senator, the restaurant serves three meals a day and sometimes even provides the evening entertainment, which has ranged from karaoke and country bands to pool tournaments, goat-roping and an ugly sweater contest.
Monte James of Yankton chose Sparky's for their "All American Breakfast" — two sausage patties, two eggs, wheat toast and homemade hashbrowns. "The food is off the charts," says James, a territory manager for Sioux Steel Company. Sioux Steel is a fourth generation family-owned business that opened in 1918 and makes grain bins, livestock equipment and other steel supplies for farmers and ranchers across the world.
James also frequents Sparky's while announcing for the Isabel Rodeo, which he has done for the last ten years. "The locals are friendly and fond of visitors," he says. " They will want to know all about your comings and goings. And as the name indicates, it is not only a grill but a bar as well and the nightlife at Sparky's is legendary." Call 605-466-2131.
Editor's Note: You can find more delicious South Dakota breakfast options in our January/February 2013 issue. To order a copy or to subscribe, call 800-456-5117.
12:26 pm - Wed, March 27 2013
Kimberley Smith said:
I would like some recipes
West River Odyssey
The Most Important Meal
Lemmon: The Cowboy Capital
Accidental Rancher