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MadArt presents "Mad Homes" a dynamic art installation in 4 old homes that are slated for demolition on Capitol Hill. 11 artists create site specific installations, and the artists have used both the interior and the exterior of the homes to express their vision.
Artists Jason Puccinelli and Liz Potter have created this wonderful trompe l'oeil on the stairs that mirrors the image on the strange and wonderful floating orbs in the living room. Enter the darkened space to become one with the art yourself and explore Manet's nude reclining Venus, "Olympia," in a whole new manner.
Interactive, complex, and an invitation to become part of the art, Liz and Jason combine audio, video, light and sound to create an immersive and profound experience that borders on magic.
Artist Ryan Molecamp has the best view of the bunch with this installation in the back house overlooking downtown Seattle and the Space Needle. His piece, called "Strain", is a large linear wall drawing depicting a river shape with structures emitting from its banks, and it wraps around the dining room and across the window. Wall sculptures of painted black wood extend outward blurring the line between interior and exterior space.
Meg Hartwig has created a sculpture using manufactured wood scraps and wood burning surface treatments, to create an imposing and slightly scary burnt wood installation.
Artist Julia Haack has created a subtle message re-using the house itself to create a symbol she believes to mean "No one at home". The diamond pattern looks organic and only upon closer examination does one realize the complexity of the creation and the integration of the piece with the main part of the house. She also did another piece entitled Keating's Foible (get out your old copy of Ayn Rand's The Fountainhead from college to reference this one), and the shape of the large imposing sculpture, created out of old lathe, is taken from architectural details from several of the houses in the Mad Homes project. Julia is known for using material from roadsides, dumpsters and building sites, and all of her current work is made with salvaged wood.
Luke Haynes has reused and recycled 100's of articles of clothing, some simple and others very unusual, to cover all the walls in one of the front houses. Like a graffiti artist, he's tagged his own work with his name right above the fireplace.
And the mad woman behind the mad dream is arts supporter and curator, Alison Milliman, the founder of MadArt and the instigator behind this and several other art projects around town. Alison has been inspired by similar projects in Australia, and vowed to bring some vibrant shows to the Seattle area upon her return. Alison and the director Brian Ohno, hand-picked each artist after a visit to their studio, and carefully planned and orchestrated this incredible installation.
Here is an interview with MadArt founder, Alison Milliman, on King FM Arts Channel.


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text: Morning all, I have posted numerous questions on here previously regarding the issue of getting music onto the HTC Desire with album art and thought it only fair to post how I have got on. I have approximately 300 CDs that I was looking to fit onto my 16gb memory card and I wanted to make sure that I chose the best method possible. I decided to use itunes to rip my music to 128kb aac, mainly due to the size issue as that should fit my collection onto the phone quite nicely with some space spare for future music. I have only ripped about 10 CDs so far but wanted to make sure I could get them onto the Desire before I ripped them all! I downloaded a program called itunes agent for my laptop, which is available from the following link: iTunes Agent I then used the guide in the following link to help me sync my desire with itunes so that all my music transferred over: How to sync the HTC Desire with iTunes - Know Your Cell Once the desire had stopped syncing I checked that the music was on the phone using the stock music player. Result the music was there with the artists and album titles all showing correctly! The only downside, no album art! It would look nice to have all my albums with the art cover showing so I checked the android market and found a great little free app called album art grabber. This found all the art instantly and now the 10 CDs are showing in the music app with art. Lastly, the music sounds great and I am well on my way to having my entire collection in my pocket!


text:
Abbott's Knowledge Of Risks Of Depakote Enough For Punitive Damages, Judge Says
(Opinion available. Document #28-160421-004Z.)
In 1999, Beth Forbes was diagnosed with bipolar disorder/manic depression. In 2003, Dr. Raziya...
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My artistic voice has been silent for so long. Maybe it has always been silenced. My psychic told me my past lives on this earth are filled with my silence. People keeping me quiet and punishing me. I am constantly being burned at the stake. This is the lifetime where my voice gets heard. At least becomes vocal, in the right ways. So far in this life it has been a process, stripping away the chains, the tape, the fear of being reprimanded. The fear of being silenced has kept me silent.
And now I am ready.
And now I know the anxiety of not being able to do art. Of not having that focus and internal connection to the divine energy. Of my energy being used and abused by another and myself. And me in turn abusing his energy. We should make it, he and I. Our artistic process simply needs to be actualized. Like the Kundalini slithering and winding up the spine, through the Chakras, shedding the old to embrace the new.
We need to shed our old habits so we can embrace the new beauty of art and understanding in our lives. And let that artistic energy be our fusion.
This is my goal for my last years in my 20's. He may not be able to endure that long. He has waited for me to have this epiphany for years now. Is it wrong of me to ask him for longer? This is a journey I have to make for myself. In order to like/love myself and life again. This journey will happen. But will he remain by my side? Will he be there in the end of the transition, joining in the artistic glory and basking in the sunlight of divine creative energy and relishing in the rewards his patience has afforded him. Rewards like stability, balance, beauty, creation, understanding, love, partnership, joy. An explosive and beautiful svadhistana chakra. Maybe even mind-blowing sex. And, materialistically speaking, a successful art career. Being able to live off the income of his talents and all that Minaxus has to offer.
I just need to do art.
Not fearing the results.
Even if the art isn't good, it will feel good making it.
And then maybe some day it will be good. And maybe someone will wan to buy a piece.
Until then. I just need to do it.
Or I will explode.
And probably get my heartbroken by the love of my life.
The man who should have no doubts or mistrusts with me. But he does b/c I am only just coming to the desperateness of this need to create. I've always known I wanted to be an artist (since I was 10 according to my psychic). No one in my family was surprised when I declared an art major. Just me apparently. And now it's time for me to accept it and do something with all that pent up energy. Haha, accept that I am a freak that doesn't belong here, as again, my psychic said. I may be misunderstood by some of those around me. He understands me though. And art understands. Maybe there is hope for this freak. Maybe there will be a happy ending. A beautiful partnership with my love in art, business, music and life.


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- dress like jess
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cancelTag what you see on the picture : clothes, celebrities, bloggers, series, movies and brands.
Originally posted 35 months ago byluxixo
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Be the first to post a tip. You will help 1 person.Do you have fashionista friends? Ask them if they know where to get these jewels!
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by Alex Billington
January 6, 2008
This question should probably have been posed as "why does January through April suck for moves" instead. There are always exceptions, usually anything in the month of March, or a few in April, like Grindhouse or Hot Fuzz, but for the most part, January and February are terrible months! They're known as the "dumping grounds" where studios drop films they know won't do well and don't have a big audience. It's always a question I've wondered, because I've realized people will always go see movies at any time. The movie-going seasons pick up around Christmas and over the summer, but throughout the rest of the year, if there's a good movie, people will always see it no matter the time of year.
The reason why this is a Sunday Discussion is because I often have to argue with people about the fact that January is a dumping grounds with terrible movies, because they just don't believe it. There is always a movie someone will end up loving in January (maybe it will be Cloverfield for me, but I'll get to that later). But for the most part, the line-up sucks. Just look at this years selections: One Missed Call, First Sunday, Mad Money, How She Move, Meet the Spartans, and Untraceable (which is the photo above) - that's it, for the entire month of January. Is there anyone who even wants to see a single one of these?
It could partially be due to the fact that there's so much else going on, that it's not really worth it to release anything big, or potentially big. Combined with the fact that everything big and possibly good was released during the always-reliable Christmas season; now it starts to maybe make sense. Beyond that you've got CES the second week, Sundance the last two weeks, and school starting back up again for students.
Last year, 300 really shook things up in Hollywood. I think for the most part studios assumed nothing would ever be that big in March and they were probably worried about its performance. Every industry analyst under-estimated its opening weekend box office, and thus when it was such an incredible success, it really changed the industry. Now that weekend, the second in March, is looked upon as a prime spot, like the "4th of July in March." However, that spot won't be as successful this year, just because there is no awesome film that audiences will love being released that weekend in March. Not at least until 2009 when Zack Snyder is back with Watchmen.
The other movie that I have a feeling could blow up into something big is Cloverfield. Universal tried a similar thing with Smokin' Aces last year. There was an excessive amount of buzz based on the trailers and it just kept building. Then they moved it to a late-January release and it did "okay," but not extraordinary at the box office. And in the end I hated it! I'm hoping Cloverfield is the exact opposite, and I have a good feeling it will be. If it is as successful as all of the buzz is promising, then it will yet again shake up the industry. A mid-January opening will never be looked upon the same again, nor will it ever be as successful again.
Things will change forever if Cloverfield does well, but besides that, January is a terrible month for movies. And why is that? It doesn't need to be, it could be filled with some fairly good films. Why does it suck for movies? At what point did the studios in Hollywood decide it was the perfect month to use as dumping grounds? And what do you think, could it ever get better or is destined to always stay this bad?
Follow Alex's main profile on :


text:
Auguste Marie François
[French oh-gyst ma-ree frahn-swa] /French oʊˈgüst maˈri frɑ̃ˈswa/ (Show IPA), 1829–1912, Belgian statesman: Nobel Peace Prize 1909.
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beeroth of the children of jaakan (Deut. 10:6). The same as Bene-jaakan (Num. 33:31).
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Clifford Whittingham [hwit-ing-uh m,, wit-] /ˈʰwɪt ɪŋ əm,, ˈwɪt-/ (Show IPA), 1876–1943, U.S. pioneer in mental hygiene. an alcoholic beverage made by brewing and fermentation from cereals, usually malted barley, and flavored with hops and the like for a slightly bitter taste. any of various beverages, whether alcoholic or not, made from roots, molasses or […]
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a city in Israel, near the N limit of the Negev desert: the southernmost city of ancient Palestine. from Dan to Beersheba. Dan (def 5). a partially reclaimed desert region and district in S Israel, bordering on the Sinai Peninsula. 4700 sq. mi. (12,173 sq. km). Capital: Beersheba. Historical Examples You have been giving me […]


text:
Hello teacher and friends, in this post I'm going to talk
about my home page. My home page is beautiful.
If you want to see my home page, click here or go to the sidebar.
But, if you want to create yours your home page click here.
It's very easy, just 25 questions.
Create your own!!!
Enjoy it.
My visit to my primary school
6 years ago


text:
- Please select a date to see the proper prices!
Description of the Accommodation
Rooms, services Bojin Business Hotel - Wuxi is offering a tastefully furnished room to those seeking accommodation in Wuxi. Since the hotel has an own restaurant, you may also try fine local meals on the spot. To learn more about the exact range of services of Bojin Business Hotel - Wuxi (including services for an extra fee) and the exact equipment of the rooms, please check the information sheet below.
Payment For the reservation you may need to provide advance payment by credit card or credit card guarantee, depending on the exact date, room and price.
Arrival, opening hours The personnel speaks Chinese - China and English.
Bojin Business Hotel - Wuxi rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Own restaurant
Desk in the room


text:
Nitric oxide synthase-2 (NOS2) is responsible for high-output nitric oxide production important in renal inflammation and injury. Using a yeast two-hybrid assay, we identified Rac2, a Rho GTPase member, as a NOS2-interacting protein. NOS2 and Rac2 proteins coimmunoprecipitated from activated RAW 264.7 macrophages. The two proteins colocalized in an intracellular compartment of these cells. Glutathione-S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygenase domain was necessary and sufficient for the interaction. [35S]methionine-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, calmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (∼60%) and NOS2 activity (∼45%) without measurably affecting NOS2 protein abundance and led to a redistribution of NOS2 to a high-speed Triton X-100-insoluble fraction. We conclude that Rac1 and Rac2 physically interact with NOS2 in activated macrophages and that the interaction with Rac2 correlates with a posttranslational stimulation of NOS2 activity and likely its spatial redistribution within the cell.
- inducible nitric oxide synthase
- guanosine 5′-triphosphatase
- protein-protein interaction
- lipopolysaccharide
- phagocyte
the free radical nitric oxide (NO) plays critical roles in numerous physiological and pathophysiological processes. In the kidney, NOS2-derived NO appears to play important roles in the evolution of ischemic acute renal failure, glomerulonephritis, and acute tubulointerstitial nephritis (see Ref. 22 for review). NO is synthesized froml-arginine by the family of NO synthases (NOS). The three types of NOS share a bidomain structure consisting of an NH2-terminal oxygenase domain, which binds heme, tetrahydrobiopterin, and l-arginine, and a COOH-terminal reductase domain that binds FAD, flavin adenine mononucleotide (FMN), and NADPH (40). NOS1 and NOS3 are basally expressed in selected tissues and are principally activated by intracellular Ca2+ transients that promote calmodulin binding sufficiently to produce low amounts of NO for signaling purposes (4). In contrast, expression of NOS2 generally requires provocation with immunoactive agents, such as bacterial lipopolysaccharide (LPS) and/or certain cytokines that activate de novo synthesis of the enzyme. Once formed, NOS2 sustains catalysis because calmodulin is tightly bound even at resting levels of intracellular Ca2+. In humans, NOS2 expression is most readily detected in monocytes or macrophages from patients with infectious and/or inflammatory diseases (31, 49). The large amounts of NO generated by NOS2 have been implicated in inflammation, host defense, tissue injury, and the profound hypotension accompanying sepsis (22, 27).
Because of its importance in cell injury and inflammation, the mechanisms governing the biosynthesis and function of NOS2 have been the subject of intense investigation. A complex array of transcriptional and posttranscriptional regulatory controls on NOS2 expression and activity have been described, including influences on transactivation of the NOS2 gene, NOS2 mRNA stability, substrate and cofactor binding and availability, and dimerization (27). For NOS1 and NOS3, interactions with heterologous proteins in addition to calmodulin have been shown to influence the activity or intracellular distribution of these isoenzymes. For example, a 10-kDa peptide, protein inhibitor of neuronal NOS (PIN), was shown to bind and inhibit the activity of NOS1 in vitro (18). Similarly, caveolins-1 and -3 interact directly with NOS1 and NOS3 (19) and inhibit NOS3 activity (19). NOS1 has been shown to interact physically with several proteins bearing PDZ domains that influence NOS1 targeting to discrete membrane domains of excitable tissues (5, 6). In the case of NOS2, thus far kalirin (34) and NOS-associated protein-110 (35) have been bound to interact with NOS2 and inhibit its activity.
In the present study, we determined that Rac1 and Rac2, members of the Rho GTPase family, physically interact with NOS2. The Rac isoforms play central roles in several important cellular functions including cytoskeletal reorganization, gene expression, and assembly and activation of the phagocytic and nonphagocytic NADPH oxidase. We further demonstrate that the point of interaction for Rac2 is the NOS2 oxygenase domain and that overexpression of Rac2 augments NO production in immune-activated murine RAW 264.7 macrophage cells. This remarkable convergence of two pluripotent signaling pathways reveals the unexpected complexity of NOS2 regulation, identifies a new target protein for Rac1 and Rac2, and suggests the possibility of coordinate regulation of the NADPH oxidase and NO production in phagocytes.
l-Glutamine, heat-inactivated FBS, penicillin-streptomycin, DMEM, and LipofectAMINE PLUS reagent were from Life Technologies. DMEM lacking phenol red, LPS from Escherichia coli O111:B4 and protease inhibitor cocktail were from Sigma. Glutathione-Sepharose 4B beads and RediPack columns, pGEX-3X, pGEX-5X, factor Xa, ECL reagents, and recombinant glutathione-S-transferase (GST) were from Amersham Pharmacia Biotech (Piscataway, NJ). Radiochemicals were purchased from Amersham. RNAzol II was acquired from TEL-TEST "B," The MATCHMAKER Two-Hybrid System 2, the Mouse Kidney MATCHMAKER cDNA library, the GAL4-activation domain hybrid cloning vector pGAD10, and pAS2–1 (GAL4-DNA binding domain hybrid cloning vector) were purchased from Clontech. Zeocin and pcDNA3.1/Zeo were acquired from Invitrogen. Mouse monoclonal anti-NOS2 antibody, anti-nitrotyrosine antibody, and an anti-nitrotyrosine-positive control were obtained from Transduction Laboratories.N G-monomethyl-l-arginine (l-NMMA) was purchased from Alexis Biochemicals. Mouse monoclonal anti-nitrotyrosine antibody was from Zymed (San Francisco, CA). Rabbit polyclonal IgGs specific for Rac1 and for Rac2 and protein A/G PLUS-agarose were purchased from Santa Cruz Biotechnologies. Oligonucleotides were custom synthesized by Genosys, the BCA protein assay kit was from Pierce Chemical, and the LipofectAMINE PLUS reagent was purchased from Promega.
Plasmids and constructs.
A fusion of a segment of murine NOS2 with the GAL4-DNA binding domain of pAS2–1 was constructed by subcloning anNcoI-NcoI fragment of the NOS2 cDNA (encoding M325-H934) into these restriction sites of pAS2–1 to yield the recombinant molecule pAS2-NOS2325–934. An expression plasmid for full-length NOS2 cloned into pCDNA3.1/Neo (Invitrogen) has been previously described (23). To generate fusions with GST, cDNA inserts encoding murine Rac2, human Rac1 (a gift from Dr. J. David Lambeth, Emory University), or various portions of murine NOS2 (M1-L1144, M1-E498, K499-L1144) were PCR amplified from cloned cDNAs or, in the case of Rac2, cDNA prepared from LPS-treated RAW 264.7 cells. The cDNAs were subcloned into convenient restriction sites in pGEX-3X or pGEX-5X vectors to maintain appropriate reading frames. For construction of a Rac2 expression plasmid, the entire coding region of Rac2 (nucleotides 35–614, GenBank Accession No. X53247) was PCR amplified from RAW 264.7 cell cDNA by using primers based on the published sequence. The Rac2 cDNA was subcloned into theBamH1 and EcoR1 sites of the mammalian expression vector pcDNA3.1/Zeo to create pcDNA3.1/Rac2-Zeo. All constructs were analyzed by DNA sequencing to confirm their authenticity and reading frames within the vectors.
Yeast two-hybrid assay.
The MATCHMAKER Two-Hybrid System 2 and the mouse kidney MATCHMAKER cDNA library were used to identify proteins interacting with NOS2.Saccharomyces cerevisiae strain CG-1945, which is auxotrophic for tryptophan and leucine and contains lacZ andHIS3 reporter genes, was grown at 30°C in either YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or minimal synthetic dropout (SD) medium (0.5% yeast nitrogen base without amino acids, 2% glucose, and 1% desired amino acid dropout solution). pAS2-NOS2325–934 and pGAD10-kidney cDNA library fusions were introduced into the CG-1945 yeast reporter strain by the lithium acetate transformation procedure (2). Cotransformants were grown on SD plates containing 5 mM 3-amino-1,2,4-triazole (to reduce background from "leaky"HIS3 expression) and lacking histidine, tryptophan, and leucine to select for cotransformants in which the pAS2–1- and pGAD10-hybrid proteins interact. The resultant colonies were then screened for β-galactosidase activity by a colony lift filter assay (2). Insert cDNAs of clones demonstrating activation of the HIS3 and lacZ marker genes were amplified by PCR of total yeast DNA by using flanking primer sequences derived from pGAD10. Selected clones were then sequenced on both strands by using an automated method. In control experiments, the GAL4-DNA binding domain and GAL4-activation domain fusion constructs were confirmed not to activate reporter gene transcription by themselves, and GAL4-activation domain fusion constructs were confirmed not to activate transcription when combined with the unrelated pLAM 5′-1, which encodes a GAL4-DNA binding domain-human lamin C hybrid in pAS2–1.
RAW 264.7 cells were incubated with basal medium or stimulated with LPS for 16 h to induce NOS2 expression. Lysates of the cells were prepared with ice-cold RIPA buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 3% protease inhibitor cocktail). One milliliter of lysate was precleared with 10 μl normal rabbit serum together with 20 μl protein A/G PLUS-agarose for 1 h at 4°C. The lysates were centrifuged at 1,500 rpm at 4°C for 5 min to remove insoluble material. Two hundred and fifty microliters of the resulting supernatant were subjected to immunoprecipitation with anti-NOS2 mouse monoclonal antibody (2.5 μg; or with an equal amount of mouse IgG) or with rabbit polyclonal anti-Rac2 (1 μg; or with an equal amount of rabbit IgG) for 1 h at 4°C together with 20 μl protein A/G PLUS-agarose for 2 h at 4°C. The immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C, and washed four times with 1 ml RIPA buffer. Proteins precipitated by anti-NOS2 or mouse IgG were separated by SDS-PAGE, electroblotted to Hybond-ECL membranes, and immunoblotted with anti-Rac2. Proteins precipitated by anti-Rac2 or rabbit IgG were immunoblotted with anti-NOS2. Bound antibody was visualized by the ECL chemiluminescent detection system, using sheep anti-mouse (for NOS2 and mouse IgG) or goat anti-rabbit (for Rac2 and rabbit IgG) IgG-peroxidase conjugate and autoradiography. The relative amount of NOS2 and Rac2 coprecipitated was grossly estimated by running 50- and 100-μg aliquots of the total lysate (not subjected to the immunoprecipitation procedure) on the final immunoblot of the coprecipitates and comparing the relative abundance of the bands.
In vitro translation.
NOS2 was transcribed and translated from pCDNA3.1/Neo-NOS2 in the presence of [35S]methionine by using T7 RNA polymerase and a troponin T-coupled reticulocyte lysate kit (Promega) by methods previously described (24).
GST pull-down assays.
Three GST-fusion proteins, constructed to contain full-length NOS2 (M1-L1144), the isolated oxygenase domain (M1-E498), or the isolated reductase domain (K499-L1144), were purified from sonicates of isopropyl β-d-thiogalactoside-induced HB101 bacterial cells according to the manufacturer's (Amersham Pharmacia Biotech) instructions. For in vitro binding assays using cell lysates, ∼4 μg of GST-fusion protein constructs were added to RediPack glutathione-Sepharose 4B columns. The columns were washed with 20 ml ice-cold PBS. Two hundred microliters of total cell lysates of LPS-treated RAW 264.7 cells prepared in RIPA buffer as above were used as a source of NOS2 protein and added to the column, followed by incubation with continuous rotation for 90 min at 4°C. The columns were then washed with 30 ml of ice-cold PBS containing 0.1% Tween followed by a final 3-ml wash of PBS, with eluates collected after each wash for analysis by SDS-PAGE. Factor Xa (50 μl in 450 μl of PBS containing 0.5% SDS) was added to the column for an overnight incubation at room temperature to cleave the bait and prey proteins from the GST tag. The final washes and the factor Xa-cleaved eluates were analyzed by SDS-PAGE and immunoblotting with anti-Rac1 or anti-Rac2 antibodies.
Studies of the direct nature and nucleotide dependence of the NOS2-Rac2 interaction were performed following the method of Spaargaren and Bischoff (39). Approximately 4 μg of GST, GST-Rac1, or GST-Rac2 were coupled to 0.1 ml glutathione-Sepharose 4B beads for 1 h at 4°C in nucleotide binding buffer [NBB; 20 mM Tris · HCl, pH 7.5/100 mM NaCl/1 mM dithiothreitol/0.1% Triton X-100/1 mM protease inhibitor cocktail] containing 10 mM EDTA overnight at 4°C to achieve nucleotide depletion. The amount of fusion protein coupled to the beads was estimated by comparing an aliquot of the slurry containing the bound fusion proteins along with 1 or 5 μg purified GST on Coomassie brilliant blue-stained SDS-PAGE gels. After 3 washes in NBB, the proteins were loaded with either GTPγS or GDPβS (Sigma) by incubation in 0.5 mM nucleotide in NBB containing 10 mM MgCl2 for 1 h at room temperature. Fusion proteins to be maintained nucleotide free were incubated in NBB containing 10 mM EDTA. The protein-bead samples were washed three times and then resuspended in NBB and either 10 mM MgCl2 (for GTPγS- or GDPβS-loaded GTPases) or 10 mM EDTA (for nucleotide-free GTPases). GST or GST-Rac2 were incubated with 30 μl of [35S]methionine-labeled full-length NOS2 translation product for 1.5 h at 4°C. The samples were then washed four times in NBB. The samples were boiled in Laemmli sample buffer and analyzed by SDS-PAGE and fluorography.
Cell culture and transfection.
The murine macrophage-like cell line RAW 264.7 was grown in complete medium (DMEM supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 10% FBS) at 37°C in a humidified incubator with 5% CO2. LPS (5 or 10 μg/ml) with or without interferon (IFN)-γ (200 U/ml) was added to the cells as indicated in the text and figure legends. For nitrite assays, DMEM lacking phenol red was used to formulate the complete medium. For stable transfections, plasmid preparations were made by using an endotoxin-free Plasmid Maxi prep kit (Qiagen). Subconfluent RAW 264.7 cells were transfected with 4.5 μg/ml pcDNA3.1/Zeo (as a vector control) or pcDNA3.1/Rac2-Zeo with the LipofectAMINE PLUS reagent according to the manufacturer's protocol to yield RAW-Zeo and RAW-Rac2 cell lines, respectively. Forty-eight hours after transfection, selection was initiated with complete medium containing 300 μg/ml zeocin. The zeocin-containing medium was replaced every 3–4 days until individual resistant colonies were isolated and established in culture as individual lines. Because RAW 264.7 cells endogenously express Rac2, the RAW-Rac2 lines were screened for successful expression of vector-derived Rac2 mRNA by Northern analysis by using a32P-labeled StuI-PvuII fragment of pcDNA3.1/Rac2-Zeo (nucleotides 525–614 of Rac2 and nucleotides 951–1271 of pcDNA3.1/Zeo) that hybridizes to vector-derived, but not endogenous, Rac2. Six representative lines of the highest expressing RAW-Rac2 clones as well as the RAW-Zeo clones were maintained in zeocin-containing medium, frozen after one to three in vitro passages, and studied in further detail.
Deconvolution immunofluorescence microsopy.
RAW 264.7, RAW-Zeo, and RAW-Rac2 cells grown on glass coverslips were stimulated with LPS for 16 h to induce NOS2 expression. The cells were fixed in buffered 3.7% formaldehyde for 20 min, permeabilized with PBS containing 0.5% Triton X-100, and blocked in PBS containing 10% goat serum at 37°C for 1 h. The cells were incubated for 1 h with anti-Rac2 and anti-NOS2 antibodies together with FITC-conjugated phalloidin diluted in PBS+10% normal goat serum at 37°C for 1 h. As a negative control, primary antibody was omitted from the immunostaining procedure. After 3 washes in PBS containing 0.05% Tween-20, the cells were incubated with Cy5-congugated goat anti-rabbit IgG (Molecular Probes) and Texas red-conjugated goat anti-mouse IgG (Molecular Probes) diluted 1:500 in PBS containing 10% normal goat serum and 0.05% Tween 20. After final washes in PBS containing 0.05% Tween, the cells were overlaid with Elvanol (DuPont) and mounted. The cells were then imaged using an Olympus IX70 inverted epifluorescence microscope. Data sets were acquired by using a mercury short-arc lamp and stored in digital format by using a cooled charge-coupled device (CCD) camera (Applied Precision, Delta Vision System). The data sets were then transferred to a Silicon Graphics workstation for deconvolution and three-dimensional reconstruction (9, 28). Delta Vision System SoftWoRx (Applied Precision, Issaquah, WA) software was used to deconvolve 0.1-μm optical sections before reconstruction. The data sets were then imported into Imaris 3 (Bitplane, Zurich, Switzerland) for digital image restoration and shadowing. For each cell, a series of 30–50 individual optical sections of 100-nm thickness in the vertical axis was obtained. Colors were arbitrarily computer-assigned to individual fluors (Cy5 = green, Texas red = red; FITC = blue).
Nitrite assays.
Nitrite, the stable metabolite of NO, was measured in culture supernatants by a modification of the Griess reaction as previously detailed (30). Standard curves were generated from sodium nitrite. Triplicate determinations were performed for each condition and represent a single observation (n). The data were normalized to protein content as determined by the BCA assay.
NOS activity assay.
Total cell homogenates were prepared at 4°C in homogenization buffer (25 mM Tris · HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA). The NOS activity of the lysates was measured for 30 min at room temperature by the formation of l-[3H]citrulline froml-[3H]arginine (30 mM) by using the components of a NOSdetect Assay Kit. Parametric studies indicated that these conditions were within the linear range of the reaction. The reaction mixture contained 25 mM Tris · HCl (pH 7.4), 3 mM tetrahydrobiopterin, 25 nM calmodulin, 0.6 mM CaCl2, 1 μM FAD, 1 μM FMN, and 1 mM NADPH, with or without 1 mM N G-nitro-l-arginine.l-citrulline was eluted from the resin supplied with the kit, and l-[3H]citrulline was quantified by liquid scintillation counting. The data were normalized to protein content as determined by the BCA assay.
Superoxide anion assays.
Superoxide anion (O ) produced from the RAW-Rac2 and Raw-Zeo cell lines was measured by using a LumiMax Superoxide Anion Detection Kit (Stratagene) according to the manufacturer's protocol. The cells were incubated with basal medium or stimulated with LPS and IFN-γ for 16 h at 37°C. The cells were then collected, and 106 cells were pelleted and resuspended in 100 μl superoxide anion medium (Stratagene). Luminol and the proprietary enhancer were added to a final concentration of 100 and 125 μM, respectively. The luminescence, indicative of the superoxide anion produced, was measured in a Turner Systems 20/20 luminometer. The data were normalized to the protein content of the samples.
Isolation of total RNA and RT-PCR for NOS1 and NOS3 was performed as described in previous work from our laboratory (29). For amplification of NOS1, primers corresponded to nucleotides 2462–2616 (forward) and 3037–3062 (reverse) of the rat sequence (3). NOS3 amplification was performed with primers directed to nucleotides 1889–1902 (forward) and 2594–2616 (reverse) of the murine sequence (13).
Triton X-100 fractionation.
RAW-Rac2 and RAW-Zeo clonal cell lines grown on 100-mm2culture dishes and treated with vehicle or LPS for 16 h were washed twice with ice-cold PBS and lysed for 10 min on ice in lysis buffer [125 mM NaCl, 25 mM Tris · HCl (pH 8.0), 10 mM EDTA, 10 mM NaF, 1% Triton X-100, protease inhibitor cocktail] or cytoskeleton-stabilizing buffer (20 mM Tris · HCl, pH 7.4, 3 mM MgCl2, 8% sucrose, 0.5% Triton X-100, protease inhibitor cocktail) (47). A "total lysate" sample was taken and, after boiling in Laemmli sample buffer, analyzed on Western blots. All fractionation steps were performed at 4°C. The remaining Triton X-100 lysate was centrifuged at 15,000 g for 10 min. The resulting pellet was washed once in lysis or cytoskeleton-stabilizing buffer, as appropriate, and taken as the "low-speed Triton X-100-insoluble fraction." The supernatant was collected and centrifuged at 200,000g for 100 min. The resulting pellet, which was washed once with lysis buffer or cytoskeleton-stabilizing buffer, as appropriate, represents the "high-speed Triton X-100-insoluble fraction," whereas the supernatant represents the "high-speed soluble fraction." For analysis by SDS-PAGE and Western blotting, all samples were boiled in Laemmli sample buffer.
Immunoblotting for nitrosylated proteins.
Immunoblots of cell lysates were prepared from RAW-Zeo and RAW-Rac2 cells that had been treated with LPS+IFN-γ overnight as previously described (14). A commercially available positive control containing nitrated proteins was used. As an additional positive control, lysates of RAW 264.7 cells that had been exposed 20 times to exogenously applied 5 mM peroxynitrite (a gift from Drs. Yasu Irie and Ferid Murad) were used. The blots were probed with two different anti-nitrotyrosine antibodies and sheep anti-mouse or goat anti-rabbit IgG-peroxidase conjugate as appropriate.
NOS2 interacts with Rac1 and Rac2 in vivo.
To identify proteins that physically interact with NOS2, a yeast two-hybrid screen was performed. A fusion gene, pAS2-NOS2325–934, containing the GAL4-DNA binding domain of pAS2–1 fused to cDNA encoding M325-H934 of murine NOS2, was constructed. Yeast CG-1945 cells expressing this construct were used to screen a library of GAL4-activation domain-tagged mouse kidney cDNA. Of the ∼3,000,000 clones screened, 9 transformants exhibited pAS2-NOS2325–934-dependent activation of both theHIS3 and lacZ markers in CG-1945 cells. Sequence analysis of one of the nine pAS2-NOS2325–934-derived clones revealed it to contain nucleotides 363–899 of murine Rac2 in-frame with the GAL 4-activation domain sequence. This sequence of Rac2 corresponds to the coding sequence beginning at codon 110 and extending into the 3′-untranslated region.
To confirm the results of the two-hybrid experiments by an independent biochemical method and to establish the occurrence of the Rac2-NOS2 interaction in mammalian phagocytic cells, a coimmunoprecipitation strategy was used. RAW 264.7 macrophage cells were treated with LPS for 16 h to induce expression of NOS2. Cell lysates were then prepared and immunoprecipitated with either anti-Rac2 or anti-NOS2 antibodies. Proteins precipitated by anti-Rac2 were separated by gel electrophoresis, transferred to nylon membranes, and immunoblotted with anti-NOS2. In the reciprocal experiment, anti-NOS2 immunoprecipitates were immunoblotted with anti-Rac2. Coprecipitation of the two proteins was taken as evidence for a direct and stable interaction between them. Figure 1 A shows the results of the coimmunoprecipitation experiments. Immunoblots of the anti-Rac2 immunoprecipitates with the anti-NOS2 antibody revealed the expected ∼130 NOS2 monomer. Similarly, immunoblots of the anti-NOS2 immunoprecipitates probed with the anti-Rac2 antibody revealed the presence of the ∼21 kD Rac2 protein. A gross comparison of the relative abundance of the immunoprecipitated proteins vs. the input amount revealed that the association appeared to be roughly stoichiometric in that ∼20% of the total NOS2 and Rac2 was coimmunoprecipitated. Because some of the protein A/G PLUS-agarose is lost in the multiple washes during the immunoprecipitation procedure, this value probably underestimates the true amount of associated proteins. In both cases, immunoprecipitation with non-immune IgG failed to precipitate the Rac2 or NOS2 proteins (Fig. 1 B). As an additional control, immunoprecipitation with the anti-Rac2 and blotting with anti-NOS2 failed to detect NOS2 in RAW 264.7 cells not pretreated with LPS (Fig. 1 C). These results confirmed the association of full-length Rac2 with NOS2 in LPS-stimulated RAW 264.7 cells.
Because Rac2 bears ∼94% structural identity to Rac1, we sought to determine whether interactions between Rac1 and NOS2 also occur in RAW 264.7 cells. Using a GST pull-down assay, lysates of LPS-treated RAW 264.7 cells were incubated with a GST-NOS2 fusion protein immobilized on glutathione-Sepharose 4B columns. After extensive washing of the columns, Rac1 and Rac2 were detected by immunoblotting of the factor Xa-cleaved samples (Fig. 1 D). These results indicate that both Rac isoforms can associate with NOS2 in these cells.
The NOS2-Rac2 interaction in vitro is direct, GTP independent, and does not require calmodulin, NOS substrates, or cofactors.
In the yeast two-hybrid system and coimmunoprecipitation assays, protein-protein interactions may be direct or mediated by a bridging endogenous protein. Therefore, Rac2 was produced and purified as a GST-fusion protein, and [35S]methionine-labeled NOS2 was translated in vitro to test their ability to bind directly in vitro in a GST pull-down assay conducted in the absence of calmodulin or NOS substrates or cofactors. In addition, to test the GTP dependence of the interaction, the GST-Rac2 fusion proteins were prebound with GDPβS, the nonhydrolyzable GTPγS, or were generated nominally free of nucleotides and subjected to the pull-down assay. As shown in Fig.2, [35S]methionine-labeled NOS2 directly interacted with GST-Rac2, but not GST alone, under each of these conditions.
The NH2-terminal oxygenase domain of NOS2 is necessary and sufficient to mediate the association of NOS2 with Rac2.
To determine the regions of NOS2 to which Rac2 binds, GST-fusion proteins encoding the isolated oxygenase and reductase domains of NOS2 as well as full-length NOS2 were used in a GST pull-down assay with lysates of LPS-treated RAW 264.7 cells. As shown in Fig.3, Rac2 in the lysates bound to the GST-full-length NOS2 fusion protein (in agreement with the experiment in Fig. 1 B) and the GST-NOS2 oxygenase domain fusion protein. However, no interaction with Rac2 was observed with the GST-NOS2 reductase domain fusion protein (Fig. 3). Because the NOS2 oxygenase construct lacks the binding sites for calmodulin and flavin nucleotides, this result indicates that the latter are not required for the interaction with Rac2. This result substantiates the findings presented in Fig. 2. The fact that Rac2 did not interact with the GST-NOS2 reductase domain fusion protein also demonstrates specificity of the interactions observed with the GST-full-length NOS2 fusion protein and the GST-NOS2 oxygenase domain fusion protein.
Overexpression of Rac2 stimulates NOS2 activity.
To determine whether Rac2 might influence NOS2 expression or activity, we stably transfected RAW 264.7 cells with a Rac2 expression plasmid or the parent vector pcDNA3.1/Zeo. To confirm that the transfection procedure did not somehow result in de novo expression of NOS1 or NOS3 in the resultant cell lines, RT-PCR analysis for these isoforms was performed by using isoform-specific primers and RNAs prepared from normal and LPS-treated wild-type RAW-Zeo cells, RAW-Rac2 cells, and mouse kidney (as a positive control). NOS1 and NOS3 transcripts were successfully amplified from mouse kidney RNA, but not from the cell lines (data not shown), indicating that NOS2 is the sole NOS isoform expressed in the transfected cell lines treated with LPS.
The RAW-Rac2 and RAW-Zeo cell lines were then examined for their ability to produce NO and NOS2 protein under basal conditions and in response to LPS. Under basal conditions, both cell lines produced comparable, but negligible, nitrite (Fig.4 A), indicating that overexpressed Rac2, in the absence of immune stimuli, cannot provoke NO generation. Figure 4 A shows that LPS promoted, as expected, high-level nitrite production in the RAW-Zeo cells, but even greater (∼60%) nitrite levels were observed in the RAW-Rac2 cells. Because nitrite production from the intact cell may be influenced by changes in the availability of NOS2 cofactors or substrate, the NOS2 activity of RAW-Zeo and RAW-Rac2 cell homogenates was measured by the l-[3H]arginine tol-[3H]citrulline conversion assay in the presence of fixed and saturating concentrations of cofactors and substrates. In response to LPS, the RAW-Rac2 cells exhibited NOS2 activity that was roughly 45% higher than that of the RAW-Zeo cells (Fig. 4 B), in close agreement with the nitrite production of the intact cells. The enhanced LPS-induced nitrite production and NOS2 activity of the RAW-Rac2 cells appeared to be principally the result of a posttranslational event, because immunoblots of lysates prepared from the cells revealed NOS2 protein amounts comparable to that of the LPS-treated RAW-Zeo cells (Fig. 4 C).
NOS2 and Rac2 colocalize in RAW 264.7 cells.
To determine whether NOS2 and Rac2 colocalize in macrophages, the subcellular distribution of NOS2 and Rac2 was analyzed by immunofluorescence deconvolution microscopy (9, 28) of LPS-treated RAW 264.7 cells. Both NOS2 and Rac2 immunofluorescence were found in a perinuclear location (judged by DAPI staining of the nuclei; not shown) as well as in more peripheral cytoplasmic sites. An overlapping subcellular localization was observed between a pool of NOS2 and Rac2 (Fig. 5). In control experiments, no staining was evident in the absence of primary antibody for either NOS2 or Rac2 (not shown).
Overexpression of Rac2 promotes NOS2 translocation to a high-speed Triton X-100-insoluble fraction.
Because Rac2 has been shown to influence the organization of the cytoskeleton and the spatial distribution of NADPH oxidase components in activated phagocytes, we hypothesized that overexpression of Rac2 might influence the subcellular distribution of NOS2 and thereby provide a molecular basis for observations of soluble and particulate forms of NOS2. As an initial approach to this question, we used Triton X-100 fractionation of lysates from RAW-Zeo and RAW-Rac2 cell lines to examine this possibility. This method has been commonly used to study Rac association with the components of the NADPH oxidase. Two separate buffer systems, lysis buffer and cytoskeleton-stabilizing buffer, were used. The results below were comparable regardless of the buffer system used to prepare the lysates. The low-speed Triton X-100-insoluble pellet has been reported to contain nuclei, large cytoskeletal networks, and caveolae. The high-speed Triton X-100-insoluble pellet contains submembranous cytoskeleton complexes and associated signaling molecules (12, 42), including Rac2 (10). In agreement with these studies, Rac2 was found in the high-speed Trixon X-100-insoluble pellet (and the low-speed supernatant from which the high-speed fractions were derived) but not high-speed supernatant in control and LPS-treated (16 h) RAW-Zeo and RAW-Rac2 cells (Fig.6 A). Rac2 was also consistently more abundant in the LPS-treated cells. NOS2 was undetectable under basal conditions in RAW-Zeo and RAW-Rac2 cell lines (Fig. 6 A). After LPS treatment for 16 h, NOS2 was apparent in the low-speed Triton X-100-soluble fraction and both the high-speed Triton X-100-soluble and -insoluble fractions (Fig.6 B). Similar results were obtained in untransfected RAW 264.7 cells after LPS treatment (data not shown). In the LPS-treated RAW-Rac2 cells, however, NOS2 was detected exclusively in the low-speed Triton X-100-soluble fraction and the high-speed Triton X-100-insoluble fraction derived from it (Fig. 6 B). These results indicate that overexpression of Rac2 promotes NOS2 translocation to and/or retention in the high-speed Triton X-100-insoluble fraction. However, deconvolution immunofluorescence microscopy, which has comparable resolution to confocal microscopy, did not demonstrate an unequivocal difference in the subcellular labeling of the NOS2 and Rac2 proteins in the LPS-treated RAW-Zeo and RAW-Rac2 cell lines (data not shown).
Effects of overexpression of Rac2 on superoxide anion production.
To assess whether overexpression of Rac2 alters superoxide anion production in macrophages, a chemiluminescence assay was used to measure superoxide anion produced by resting or LPS+IFN-γ-stimulated RAW-Zeo and RAW-Rac2 cells. As shown in Table1, RAW-Rac2 cells generated greater levels of superoxide anion than RAW-Zeo cells, suggesting, for the first time, that Rac2 overexpression drives superoxide production by the NADPH oxidase. LPS+IFN-γ treatment resulted in a significant reduction in the amount of superoxide anion produced by both groups of cell lines. Because LPS+IFN-γ treatment promotes NO production in these cells, and because NO chemically inactivates superoxide anion by forming peroxynitrite, we reasoned that the lower levels of superoxide anion in the activated RAW-Rac2 and RAW-Zeo cell lines might reflect a quenching of superoxide by NO. Accordingly, we assayed superoxide production in LPS+IFN-γ-treated cells when NOS2 was inhibited withl-NMMA. With NOS2 inhibited, superoxide anion production was enhanced in the LPS+IFN-γ-treated RAW-Zeo cells and to a far greater extent in the RAW-Rac2 cells (Table 1). If thel-NMMA-dependent component of superoxide anion production represents the superoxide anion inactivated by NO, this result suggests that the RAW-Rac2 cells form far greater levels of peroxynitrite than the RAW-Zeo cells after exposure to LPS+IFN-γ. Alternatively, high concentrations of NO peroxynitrite have been reported to inactivate the NADPH oxidase in polymorphonuclear leukocytes (26).
High concentrations of peroxynitrite are known to nitrosylate target proteins. Protein nitrosylation, therefore, can be used as a surrogate marker for peroxynitrite generation in cells. Accordingly, we probed immunoblots of lysates prepared from control and LPS+IFN-γ-treated RAW-Zeo and RAW-Rac2 cells with antibodies directed against nitrotyrosine. As positive controls, we used a commercially available panel of nitrated proteins and lysates that had been prepared from RAW 264.7 cells repetitively treated with 5 mM peroxynitrite. No remarkable difference in the intensity or pattern of protein bands was observed between the RAW-Zeo and RAW-Rac2 cells under basal or induced conditions with either of the anti-nitrotyrosine antibodies (not shown).
In this study, we report several novel findings important to an understanding of NOS2 biology, Rac1 and Rac2 signaling targets, and macrophage function. By demonstrating the physical interaction of NOS2 with Rac1 and Rac2 in activated murine macrophages, we provide new examples of heterologous proteins other than calmodulin that physically associate with NOS2 and identify NOS2 as a new target for the Rac isoforms. Our demonstration that overexpression of Rac2 augments NOS2-generated NO production and NOS activity in LPS-treated RAW 264.7 cells establishes not only the functional importance of the NOS2-Rac2 interaction but also suggests a novel posttranslational mechanism for controlling NO synthesis. In vitro binding studies showed that Rac2 binds to the oxygenase, but not the reductase domain of NOS2, that the interaction is direct, and that it can occur in vitro in the absence of calmodulin, heme, and NOS2 substrates and cofactors. Finally, our finding that NOS2 exclusively distributes to a high-speed Triton X-100-insoluble fraction in RAW 264.7 cells overexpressing Rac2 suggests that the interaction influences the spatial distribution of NOS2 in the cell, apparently in such a way that optimizes LPS-induced NO release. Given the diverse cell processes in which both NOS2 and the Rac isoforms participate, the physical association of these proteins in vivo suggests the possibility that the interaction may play important biological roles.
Rac2 is principally expressed in cells of myeloid origin, where it participates in the formation of the NADPH oxidase complex in phagocytic cells (8), and it is also believed to be a component of the NAD(P)H oxidase in vascular smooth muscle cells (33). Its close structural relative, Rac1, is more widely distributed among cell types and has been implicated in diverse cell processes, such as the organization of the actin cytoskeleton, protein kinase cascades, transcriptional activation, and cell cycle progression (15). The Rac proteins are believed to play a role in membrane ruffling and phagocytosis in macrophages. The NADPH oxidase of phagocytes generates superoxide anion and other reactive oxygen species such as H2O2, HOCl, and OH that are cytotoxic to invading pathogens (8). In resting cells, the phagocytic NADPH oxidase is composed of membrane-associated flavocytochrome b559 and several cytosolic proteins, including p47phox, p67phox, p40phox, and Rac2 or, less commonly, Rac1. When the phagocytic NADPH oxidase is activated to generate superoxide anion, p47phox, p67phox, and Rac2 translocate to a detergent-insoluble subcellular fraction (10) and become stably associated with the plasma membrane (8). Similarly, we found that overexpression of Rac2 in LPS-treated RAW 264.7 cells promotes redistribution of NOS2 from high-speed Triton X-100-soluble and -insoluble fractions exclusively to the Triton X-100-insoluble fraction. Whether Rac2, through its physical association, ushers NOS2 from the high-speed Triton X-100-soluble compartment to the high-speed Triton X-100-insoluble compartment, facilitates its retention in the latter compartment, or acts indirectly to promote NOS2 translocation from one compartment to the other will require additional study.
In suggesting that the distribution of NOS2 within the cell might be regulated and that Rac2 is involved in this process, the present data extend earlier reports of soluble and particulate forms of NOS2 (16, 17, 38, 48). Hiki and co-workers (17) demonstrated nitrite-producing activity in the high-speed pellet of sonicated peritoneal macrophages from Calmette-Guerin bacillus-treated rats. A portion of the particulate activity was Triton X-100 insoluble. Hecker et al. (16), studying IFN-γ-treated J774.2 macrophage-like cells, recovered roughly one-half of the cGMP-elevating activity in a 200,000-g pellet, but the molecular identity and detergent solubility of the activity were not established. Schmidt et al. (38) found that roughly one-third of NOS activity in LPS and IFN-γ-treated RAW 264.7 cells distributed to a 105,000-g, KCl-washed pellet. The majority of the particulate activity remained insoluble in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Finally, Vodovotz and co-workers (48), using activated primary macrophages, found approximately one-half of the NOS2 activity and immunoblotted NOS2 protein in a 100,000-g, KCl-washed pellet. Of this particulate activity, about one-half was Triton X-100 insoluble. Immunoelectron microscopy demonstrated NOS2 immunoreactivity in 50- to 80-nm vesicles that were not labeled by lysosomal or peroxisomal antibodies. Spatial restrictions of NOS2 within the cell presumably serve functional purposes. Because NO and O are both produced by activated phagocytes, production of more toxic reactive oxygen and nitrogen species, such as peroxynitrite (ONOO−), is favored with the prospect of injury not only to pathogens but also to the host cell. Compartmentalizing NO production may represent a means for the cell to optimize microbial killing or inhibition of viral replication and minimize exposure of host cell components to these toxic molecules. Whether Rac2 ushers a pool of NOS2 to the NADPH oxidase remains to be clarified. Further studies will also be needed to determine whether the NOS2-Rac2 interaction alters the GTPase activity of the Rac proteins. Furthermore the biochemical composition and ultrastructural correlate of this compartment remain to be determined, because we did not discern a distinct difference in intracellular locale at the resolution of deconvolution immunofluorescence microscopy.
It is intriguing to speculate that the association of Rac2 with both the NADPH oxidase complex and NOS2 may place superoxide anion and NO production in close proximity to facilitate the formation of peroxynitrite for microbicidal effects or, in contrast, to promote the NO-mediated inactivation of the NADPH oxidase (26) to limit damage to host tissues. We found that Rac2-overexpressing macrophages produced more superoxide anion than did controls under basal conditions, suggesting that Rac2 may be rate limiting for NADPH oxidase activity. In addition the Rac2-overexpressing cells generated little superoxide anion when NOS2 was induced by LPS+IFN-γ but high amounts when NOS2 was inhibited with l-NMMA. These data suggest that the superoxide anion produced by the LPS+IFN-γ-treated macrophages rapidly reacts with NOS2-generated NO to form peroxynitrite. However, because the relative amounts and patterns of proteins immunoreactive with two different anti-nitrotyrosine antibodies, an index of peroxynitrite formation, did not remarkably differ between the LPS+IFN-γ-treated Raw-Rac2 and the Raw-Zeo cells, it remains possible that NO inactivates the NADPH oxidase in these cells to limit superoxide anion production, as has been reported in other cell types (26).
The Rac proteins generally function as molecular switches, cycling between an active GTP-bound and an inactive GDP-bound state. In most examples, the active GTP-bound Rac binds to target effector proteins that relay cell signals. We found that recombinant Rac2 bound to NOS2 in vitro in the GDP-bound state and in the nominal absence of guanine nucleotides (Fig. 2). GTP-independent interactions of Rac isoforms in vitro have also been reported for other targets, such as Sra-1 (20), type I phosphatidylinositol-4-phosphate 5-kinase (45), and diacylglycerol kinase (44). Although we did not observe a consistent difference in the amount of in vitro translated NOS2 bound to GTP- or GDP-bound GST-Rac2 (Fig. 2), it remains possible that GTP-GDP cycling may influence the kinetics or stability of the interaction in vivo.
The fact that NOS2 interacted with both Rac1 and Rac2 in the GST pull-down assays (Fig. 1 B) suggests that NOS2 associates with regions common to the Rac isoforms. This common sequence region would include all but the extreme COOH termini of the Rac proteins. The identification of the minimal region(s) within the NOS2 oxygenase domain to which Rac1 and Rac2 binds will be important, because it will not only provide insights into NOS2 bioregulation but also may help to identify other targets for the Rac isoforms and related Rho GTPases. Because the Rho GTPases participate in numerous cell processes, considerable effort has been made to identify molecular targets of these proteins. Several protein motifs recognized by Rho GTPases have been characterized in various target proteins, including the CRIB motif (7), REM-1 (36), and POSH (41), but these motifs are absent from the oxygenase domain of NOS2. Rac2 has been shown to bind in vitro to the COOH terminus of p67phox (11) and tetratricopeptide repeat motifs in the NH2-terminal region of p67phox (21), but there is no significant homology between p67phox and NOS2. Similarly, other known Rac targets, such as PAK (43), Wiskott-Aldrich syndrome protein (37), POR1 (46), IQGAP1 (25), and specifically Rac1-associated protein (20), also share no sequence homology with NOS2. Thus the Rac binding motif in NOS2, once defined, will be novel. Further definition of the protein domains of NOS2 and Rac2 that interact will require additional studies, as will determination of whether other members of the NOS and Rho GTPase families can associate with one another. Future studies to investigate the functional and regulatory aspects of NOS2-Rac relationships should provide fresh insights into the cell biology of not only phagocytic cells but also the numerous tissues and cell types in which these proteins are expressed in physiological and pathophysiological states.
Biosynthesis of NOS2 is principally regulated at the level of gene transcription. The posttranslational control of NOS2 activity has received little investigative attention. Inferential evidence suggests that tyrosine phosphorylation of NOS2 in activated RAW 264.7 cells may increase NOS2 activity by a posttranslational mechanism. Exposure of activated RAW 264.7 cells to a tyrosine phosphatase inhibitor significantly increased the level of NOS2 tyrosine phosphorylation and NOS2 activity (32). The present report suggests a second example of a posttranslational mechanism that augments activity of the enzyme. The fact that NOS2 activity in the RAW-Rac2 cell homogenates, under conditions in which the concentrations of substrates and cofactors were fixed and saturating, was greater than that of RAW-Zeo cells suggests that Rac2 influences the activity of the enzyme via a direct mechanism rather than by increasing the availability of substrate or cofactors. This conclusion is supported by knowledge that intracellular levels of heme and tetrahydrobiopterin increase coincident with NOS2 induction in activated RAW 264.7 cells (1) and that further supplementation of these prosthetic groups or the substrate l-arginine does not increase NOS2 activity or dimerization in these cells (1). Further studies will be required to decipher the specific mechanism by which Rac2 enhances NOS2 activity.
We thank Dr. J. David Lambeth, Emory University, for the gift of human Rac1 cDNA.
This work was supported by National Institutes of Health Grants DK-50745 and GM-20529 (to B. C. Kone) and the Department of Defense "DREAMS" Center. It was conducted during Dr. Kone's tenure as an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: B. C. Kone, Div. of Renal Diseases and Hypertension, Depts. of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, The Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.138, Houston, TX 77030 (E-mail: Bruce.C.Kone{at}uth.tmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2001 the American Physiological Society


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Here are the modern 3 level house design ideas with Carara marble. this house high-tech electronic furniture design and decorating. this house with six bedrooms and seven bathrooms are bar, office, walk in pantry, gym, media room, wine cellar storage room filled room, covered patio with fireplace at home. Surrounded by palm trees and exotic fruit trees, the house is almost a tropical resort with a pool in the courtyard. This stunning exotic wood used around doors and built-ins and ending with the curved exterior walls. let's check the Stunning Modern Luxury Three Level House Design with Carara Marble picture below:
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Browse this site http://germandogtrainingcenter.com/ for more information on Sacramento Dog Training. We often hear stories about dogs not respecting children in the home or dogs being in competition with children for attention. It may come as a surprise then to learn that children can actually be involved in Sacramento Dog Training from a young age. Getting children involved in dog training will depend on several factors but it can be a great way for a child to learn about dogs. follow us : http://marysvilledogtraining.yolasite.com/
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Every German Shepherd puppy from my kennel is vaccinated, wormed, tattooed and temperament tested before leaving my kennel. My selective breeding program includes only 100% German bloodlines.
Thomas Sauerhoefer has over 30 years experience in Schutzhund Training. He has put 350 titles on dogs in Germany and America. Thomas is a specialist in Schutzhund protection work, especially problem dogs. Helping weak dogs with bite problems, and dogs with no outs. He is an expert for both show and working lines.
From a BH to Schutzhund 3, I will train your dog to achieve the best score the dog is capable of handling. If the dog fails during a trial when attempting to earn a BH or Schutzhund title, I guarantee that I will continue to work and train the dog until we pass. Your dog and I are a team when we are out on the field and I will work to the best of my ability to make sure that we both perform at our best level.
While dogs of other breeds are also admitted to Schutzhund trials, this breed evaluation test was developed specifically for the German Shepherd Dog. Schutzhund is intended to demonstrate the dog's intelligence and utility. As a working trial, Schutzhund training measures the dog's mental stability, endurance, structural efficiencies, ability to scent, willingness to work, courage and trainability.
This working dog sport offers an opportunity for dog owners to train their dog and compete with each other for recognition of both the handler's ability to train and the dog's ability to perform as required. It is a sport enjoyed by persons of varied professions, who join together in a camaraderie born of their common interest in working with their dogs. Persons of all ages and conditions of life — even those with significant disabilities — enjoy Schutzhund as a sport. Often, it is a family sport.
German imports are used in my breeding program to maintain the German standard and I continually strive to do my part to improve the breed. I raise my German Shepherd puppies with much love and care, in a home environment to ensure proper socialization and so that I can continually evaluate each of the German Shepherd Puppies that I breed. I have a lot of experience evaluating puppies and I use my experience to ensure that each puppy and new owner are a perfect match. It is important to me that both owner and Puppy are happy with each other. Each German Shepherd Puppy vom Heidelberger Schloss comes with a written health and hip guarantee, as well as guidance and advice for the life of your dog.Sacramento German Shepherd Puppies
German Shepherd Puppies make excellent family companions, protection dogs, sport dogs and can virtually perform just about any job you can think of. The German Shepherd Dog is an intelligent and noble breed, that does well in a variety of living situations. Whether you are an individual looking to buy a dog to perform as a sport companion, looking for a best friend, a family looking for a dog to watch over the kids, a personal protection dog to keep you safe; my kennel has a German Shepherd Puppy that will fulfill your every need..
Picking up your German Shepherd Puppy from the German Dog Training Center is extremely easy for those located in Northern California. The facility is easily accessible from Sacramento, San Jose, San Francisco Bay Area, Chico, and Redding; located only 45 miles North of Sacramento.
For those who live farther away, arrangements can be made to ship your German Shepherd Puppy to an airport near you. Before being shipped by plane, each German Shepherd Puppy receives a health certificate from a Veterinarian to ensure that the puppy is healthy and ready for the flight.
The German Dog Training Center is Northern California's Premier German Shepherd Personal Protection Training Facility
Having a dog that can not only be a family companion, but a protector can bring you piece of mind and security.
Personal Protection Level I – $2,500
A personal protection level I dog is trained to protect you and your family on command. The dog is trained to bark and hold and keep a threat at bay, while primarily acting as a deterrent to would be assaulters.
Personal Protection Level II – $4,500
A personal protection level II dog is trained to bite on command and release on command when handler is near the dog.
Personal Protection Level III – $6,000
A personal protection level III dog is trained to bite and release on command and can be sent out to bite and release on command from a distance.
Visit our website the German standard and I continually strive to do my part to improve the breed. I raise my German Shepherd puppies with much love and care, in a home environment to ensure proper socialization and so that I can continually evaluate each of the German Shepherd Puppies that I breed. I have a lot of experience evaluating puppies and I use my experience to ensure that each puppy and new owner are a perfect match. It is important to me that both owner and Puppy are happy with each other. Each German Shepherd Puppywww.germandogtrainingcenter.com
Sacramento German Shepherd Puppies


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This is a very rare and beautiful Victorian garnet ring set in rose gold, dating circa 1880s. Ornate setting with an elaborate floral pattern, which has been hand engraved. The band is marked 15 K. The ring features 3 lovely crimson colored garnets. , the center garnet being slightly larger than those flanking it. From one side of one garnet to the other end of third garnet, it measures 3/4". The face of the ring measures 3/8" north to south. Size 9.25. This is an English piece and would be appropriate for a man or woman. Excellent condition.
Please note, Ruby Lane's calculated shipping is an estimate and sometimes can be intimidating. Rest assured, I only charge my actual shipping costs and will refund any excess you have paid. I sell vintage and antique items with a history; oftentimes items may show wear from a former life. I always try to note any serious condition issues. Layaway is available. A 20 percent non refundable deposit will secure the item and we will work with you regarding the length of the layaway.
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|29 Dec 2010
Win 7 ultimate 64 bit
Hi Dean P,
I have not heard from you in a while, are you still going to help me?
I don't mind waiting for you, as I know that you will come back to me when ready.
The last that I heard is that you were analyzing the dumps, I hope to hear from you soon.
Kind regards
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When i start task Scheduler it say that Task BackgroundConfigSurveyor:The task image is corrupt or has been tampered with what should i do to perform a task. :cry:
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the task image is corrupt or has been tampered with
A clean installation referred to today in another thread was done by a computer repairer. As this was very recent I would not expect such warnings on viewing the task scheduler ( and they were numerous). I was not looking to seal with any particular task, just nosing! So is the scheduler...
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Last month I had the pleasure of having a chat with Michael Ford in Amsterdam. Check the results in this month's Blues In Britain magazine. A two page interview with yours truly, as Little Steve & the Big Beat will be part again of this year's P&O Blues Cruise!
Tickets UK: http://www.poferries.com/en/hull-rotterdam/minicruises/themed-minicruise/blues-revue-minicruise-to-rotterdam
Tickets NL: http://www.poferries.com/nl/rotterdam-hull/minicruise/blues-york


text: I have playlists on my phone as albums but the songs are all in the wrong order which is a problem for albums like "War of the Worlds" which is supposed to play in order of track number! Similar thing with photo's. On my iPhone I could set up different folders for holiday snaps etc but even when I create the folders directly, they all get lumped together in "camera shots." Any help appreciated. Cheers, Chris


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Item description for The Very Hungry Caterpillar (Very Books) by Eric Carle...Overview
Follows the progress of a hungry little caterpillar as he eats his way through a varied and very large quantity of food until, full at last, he forms a cocoon around himself and goes to sleep. Die-cut pages illustrate what the caterpillar ate on successive days.The beginning reader may watch a hungey caterpillar eat his way to the cocoon stage, when he hibernates for two weeks to emerge as a beautiful butterfly
Publishers Description
THE all-time classic picture book, from generation to generation, sold somewhere in the world every 30 seconds Have you shared it with a child or grandchild in "your" life?
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The Very Hungry Caterpillar (Very Books) by Eric Carle has been reviewed by professional book reviewers and journalists at the following establishments -
Promise Angels is dedicated to bringing you great books at great prices. Whether you read for entertainment, to learn, or for literacy - you will find what you want at promiseangels.com!
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Like other organ systems, such as the gastrointestinal and musculoskeletal systems, age negatively affects certain features and functions of the reproductive system.
A recent study found that feeding mares too much or too little had lasting effects on the health and development of their foals.
For years, research has focused on both getting and keeping mares in foal. New studies in human medicine suggest that measuring specific pieces of genetic material called microRNAs could help veterinarians better diagnose pregnancy-related problems.
According to a recent study, stallions can alter the composition of their semen to optimize "ejaculate economics."
Most broodmares should be maintained in moderate to moderately fleshy body condition throughout gestation. Achieving weight gain in late pregnancy is difficult for some skinny mares because so many of the calories consumed are being diverted to fetal growth.
Foals born to Thoroughbred mares are heavier now than in past years, and researchers recently set out to discover if the one-two punch of obesity and endocrine dysfunction affects birthweight.
Increasing mare age is associated with increased fertility issues, and older mares have lower foaling rates than their younger counterparts. In addition to simply being "older," underlying pituitary pars intermedia dysfunction (PPID, equine Cushing's disease) is also thought to impact reproductive function in mares.
A recent study found that wild herds contribute to the decline of native flora and fauna populations, and are thought to spread disease.
The goal of providing extra calories to pregnant mares by supplementing with concentrates from around the start of the seventh month until weaning will ensure certain mares do not become too thin.
Uterine inflammation can prevent pregnancy, and infection of the placenta later in the pregnancy can cause the mare to abort the fetus before it is fully developed.
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Ben Harper
Jenny O.
Les Schwab Amphitheater
344 SW Shevlin-Hixon Drive
Bend, OR, 97702
Doors 5:00 PM / Show 6:30 PM
Watch & Listen
Ben Harper is a musician, artist, and activist. Whether it's through the soul of southern Gospel, 70s funk, blues, reggae, or straightforward rock and roll, Harper and his trademark Weissenborn guitar have been stunning audiences with incendiary live performances and timeless songwriting.
Harper continues to innovate his craft as a songwriter, performer, and producer. He has released 10 acclaimed studio albums since 1994 – including Give Till It's Gone, which debuted at No. 15 on The Billboard 200 in 2011 and was hailed by Rolling Stone as "his most searingly personal album" – and three live records. These releases combined with an unceasing commitment to international touring have helped Harper grow a fan base that now spans several continents. After spending the summer on the road with his band, Harper will launch his first-ever acoustic headline tour in September.
A collaborator by nature, Harper has recorded and performed with a long list of artists including Ringo Starr, Pearl Jam, Jackson Browne, Tom Morello, Rickie Lee Jones, Taj Mahal, Jack Johnson and Vanessa Da Mata. His album with the Blind Boys of Alabama, There Will Be A Light, earned two GRAMMY® awards for Best Pop Instrumental Performance and Best Traditional Soul Gospel Album.
"Jenny O. just released her debut, Home EP on Manimal Vinyl. "The five-song, self-produced EP showcases Jenny O.'s flair for dreamy folk, jazz-fueled hip-hop, '60s- and '70s-style pop arrangements; the lyrics are conversational and down-to-earth, and everything seems appropriately drenched in LA sunshine." - Shockhound
"Influences ranging from jazz to the The Beatles, and a strong sense of composition across the board." - ROXY
$39 + $1 to Bend-La Pine Schools Music Programs
Tickets Available at the Door
Upcoming Events
Les Schwab Amphitheater
Fri, June 8
JETHRO TULL 50th Anniversary Tour


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I love face masques and I've mentioned this one before but thought I would do a little review of it as I've been using it for a little while now. It's from CLAYSPRAY and I chose the GLOW one which has mineral rich red clay in.
Clayspray as a brand use natural sourced mineral clay for their products and there are several different masques available suited for different skin types.
The one I chose has spring water in and cacao, it's easy to use, you can either spritz your face with the mineral hydrate h20 spray you get if you purchase any of the Clayspray masques from FEELUNIQUE. you get this free. If you haven't got the hydrate spray you can just wet your face. You squeeze a little into your palm and then rub into your face avoiding the eye area. You leave this to dry, so it goes from rich and chocolatey to dry and obviously masque like. I've photographed the process to show you how it looks when it's in it drying process. To get if off you simply rinse with lukewarm water. I normally do this when I have a bath as I think the steam helps the masque soak into your skin better. Plus it's easier to wash off in the bath. The water looks like something from Charlie and the Chocolate factory once it's rinsed off.
The results are a really clean looking face and does give you a nice natural looking glow I really like it.