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This spa hotel was part of a complex called Termalium Pools has a total of 117 rooms spread over 3 floors. It has a lobby with a reception open 24 hours, currency exchange facility, lift, cafe, kiosk, bar and restaurant. They are also available laundry service and medical assistance (charges apply) and there is a bike storage and parking.The rooms include an en suite bathroom, direct dial telephone, satellite TV, Internet connection, air conditioning and central heating as well as a terrace. Only the superior room offer double bed.
By car : Madrid N-III to Albacete, N-301 Murcia exit Archena (KM 373) - Barcelona A-7 to Murcia, N-301 Murcia-Albacete exit Archena (KM 373). By train : Archena station.

subset: commoncrawl


text: For further information or to arrange an interview with Colleen Sherin, Senior Fashion Director, please contact Heather Hertz at firstname.lastname@example.com or 212.451.3649.About Saks Fifth Avenue Saks Fifth Avenue, one of the world's preeminent specialty retailers, is renowned for its superlative American and international designer collections, its expertly edited assortment of handbags, shoes, jewelry, cosmetics and gifts, and the first-rate fashion expertise and exemplary client service of its Associates. Today, Saks operates 43 full-line stores in 20 states, 5 international licensed stores, 65 Saks Fifth Avenue OFF 5TH stores and saks.com, the company's online store. Saks Fifth Avenue is proud to be named a J.D. Power and Associates 2012 Customer Service Champion and is only one of 50 U.S. companies so named.


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This clutch kit is for the Audi 5000 MC1 2.2L Turbo motors with the stock flywheel. Clutch level descriptions: Stage 1: Stock-like daily drivability and very good wear characteristics. High clamp pressure plate, steel-backed and multi-compound woven organic material and high torque sprung hub and disc assembly. The perfect choice for a slight upgrade from stock type power levels, for example, just a chip modification. Stage 3+: This is the ultimate High HP Street kit, offers good, stock-like daily drivability and better manageability than the stage 3, with even better wear characteristics. High clamp pressure plate, full-faced carbon metallic friction material, high torque sprung hub and disc assembly. We recommend this for street or track driven applications up to 550 crank HP, you simply will not wear this clutch out.
Please Note: This clutch is a non-stock item and availability is dependent on the manufacturer's current inventory. Allow up to 48 hours for processing when ordering, and please contact our Sales Staff for current availability if a rush order is needed.
Note about break-in: This clutch uses a heavy duty clutch disc that performs well in very high torque use that produce extreme shear forces and heat. In order to gain this performance advantage, the disc friction material is much harder and more robust than the typical OEM organic disc. In addition, the disc backing plate, what the friction material is riveted to, is much more rigid. OEM clutch discs use a type of belleville spring washer to allow the disc to compress and be compliant when being clamped between the pressure plate and flywheel friction surfaces. These high performance clutches, however, use a solid steel backing plate which does not compress at all, this type of construction gives the disc its incredible strength, but means that the disc will require more break-in time than the compliant OEM counterpart. Keep in mind that this clutch may have trouble fully disengaging the first time it's used, and will require much more break-in than the OEM clutch to achieve normal use. Typically up to 1000 miles of use is normal to get full break-in. Be sure break-in use consists of generous stop and go as well as shifting, the slipping action as the clutch is engaged is what facilitates the break-in process. Ultimately the break-in process is one that allows the clutch disc to conform to the pressure plate and flywheel friction surfaces, once these surfaces conform to each other they perform very well and offer the maximum clutch holding power possible. Be patient with initial use and driving and you'll be rewarded with an incredible performing clutch system!


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My wife and I are 29 yrs. old from New York. We are foodies who will be in Vegas for three nights after spending four nights in SF/Napa. We are staying at the Palazzo, and it is my wife's first time in Vegas. We have all the celeb chefs in New York, and we want to narrow our experiences to the best food. So far, we've heard that Lotus of Siam and Raku are must experiences even though they are off the Strip. Agree/disagree? First, has anyone been to Lotus of Siam for lunch and had the buffet? Second, does anyone have a preference between SW, Stripsteak, and CUT? Third, has anyone been to Barlotta Restorante in the Wynn?
Any other thoughts or suggestions would be appreciated!
Lotus of Siam
953 E Sahara Ave Ste A5, Las Vegas, NV 89104


text:
It's taken a wee bit of detective work but it looks like LG (which has announced the LG Surf 4GB and revealed the Optimus handsets), may be on the verge of announcing a new series of mobile phones based upon the Android operating system. The evidence comes from the Intomobile website and a not particulary photogenic picture of the included User Agent profile which focuses on a new design apparently called the LG P950. Needless to say, the proposed phone has not been confirmed to exist by LG, never mind announced.
According to the UA details, the new phone will feature a WVGA screen packing in 800 x 480 pixels and will include HSDPA / HSUPA for fast data transfers. An included browser will be included based on a Webkit variant.
Other details are, for now, unavailable as is a proposed release date, distribution, price and so on. However, when we find out more, we'll pass it onto you.
Click to read the Intomobile story


text:
Erkanmatik 2013 03 by ErkanKalenderliWatch
©2013-2017 ErkanKalenderli
Love the perspective!
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Nerede çekildi bu fotoğraf?
Nerede çekildi bu fotoğraf?
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Really cute!
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I love the angle of the shot. The textures are beautiful.
Love the colors in the background. Focus is well captured on eyes. Very contrasty texture on the bark. Love it!
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What a great shot
What a great shot
Love it! Makes you want to get to know this cute animal instead of making stupid memes
A very nice shot!
good shot and I like this Animal


text:
Present-Holiday is an image song for Athena Asamiya that appears only on the King of Fighters '98 Arranged Soundtrack. It was the first song that her current voice actress had performed for the character.
| Japanese lyrics
Mazamashi wo machikirezu okita asa wa
Furisosogu hikari made biizu-mitai
Umaretate no kaze mado wo aketara torokeru
Oboetate no uta pajama kita mama de kuchi-zusamu
Jibun no tame ni okurimono sonna ichi-nichi
Karabo no kokoro ni nai wo tsukamae ni yukou kara...
Hachiue no gaabera ni mizu wo ageta
Sutoreeto no kami karuku maitara hajimaru
Ichiban suki na shiroi fuku wo kite dekakeyou
Yakusoku ni nai monogatari sonna deai wo
Ano ka domagattara kitto shiranai watashi ni naru...
Kisetsu no naka de hashaide 'ru sonna shiawase
Chiisaku akubi wo shita koneko kossori tsuite 'ku
| English translation
I couldn't wait for the alarm clock to wake me in the morning
Beads of light seemed to pour in
Through the window opened by the newborn wind
I wear pajamas just like I learned by word of mouth from a song
Like a present for myself everyday
What will I go catch with this empty heart...
As I watered the potted gerbera
The light straight hair begins to curl
I go out and wear the white clothes I like best
Meeting in a story of no promises
That corner which bends surely doesn't know it is becoming me...
Within this miracle, I will run towards happiness
The kitten secretly lets out a tiny yawn


text: Origin Agritech Limited (NASDAQ: SEED) ("Origin" or the "Company"), a vertically–integrated supplier of hybrid and genetically-modified crop seeds, announces plans to establish a joint venture company in Xinjiang for seed production and distribution. The venture will be with a prominent local, state-owned partner, Xinjiang Jinbo Seed. Facing increased demand for certain seed varieties, the company expects to spend up to US$ 15 million in investment for a 51.0% ownership stake to build an additional state-of-the-art center to produce, process, package, and sell high quality seed products for the domestic China market. With the completion of new facility, Origin will double the seed conditioning and production capacity. Furthermore, the joint venture will open up marketing channels to distribute Origin hybrid products to the northwest region in China. These additional sales are incremental to Origin's existing sales while possessing the same gross margins. Origin will possess the exclusive joint venture partnership with this local partner in Xinjiang. Xinjiang, is an emerging area in China, possesses 17% of the total land acreage of mainland China. The adjacent region also possesses distinct advantages in high quality land for the production of seed. Given the large farm size in the direct area, the farms possess modern mechanization and advanced irrigation systems. Initial production is scheduled to start by December 2011 at the central production facility, with a stepwise ramp-up to the full facility by August 2012. The proposed location will become final pending local approvals. Upon full operation the facility is designed to allow 50,000 tons of seed production capacity. "Origin is experiencing strong domestic demand in certain seed products because of its technical leadership and we need to increase our production capacity and flexibility," said President and Chief Executive Officer Liang Yuan, adding that the plant will "diversify the company's production base to meet the needs of a changing cost structure and enhance the company's consistent seed quality, in addition to expanding our sales and marketing base in the region."


text:
|Publication number
|Publication type
|Application number
|Publication date
|Jul 4, 1989
|Filing date
|Dec 17, 1986
|Priority date
|May 21, 1984
|Fee status
|Publication number
|David L. Urdal, Carl J. March, Steven K. Dower
|Original Assignee
|Immunex Corporation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (2), Non-Patent Citations (37), Referenced by (28), Classifications (14), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of U.S. patent application Ser. No. 825,360, filed Feb. 2, 1986, now abandoned, which is a continuation of U.S. patent appllication Ser. No. 612,559, filed May 21, 1984, now U.S. Pat. No. 4,578,335.
The present invention relates to interleukin 2 receptor, and more particularly to a hybridoma which secretes a monoclonal antibody capable of binding to interleukin 2 receptor.
Many normal immune responses require participation by T-cells. T-cell proliferation is controlled by the presence of a polypeptide growth hormone, interleukin 2 (hereinafter IL-2), formerly known as T-cell growth factor (TCGF). See Gillis and Smith, Nature 28: 154 (1977). The mechanism by which IL-2 controls T-cell growth is not completely understood. However, it is known that IL-2 acts on T-cells via a specific, high affinity plasma membrane receptor. In order to continue to divide, IL-2 dependent T-cells must express this receptor and IL-2 must bind to a portion of this receptor. Robb, et al., J. Exp. Med. 154: 1455 (1981). A more complete knowledge of the biochemistry of the IL-2 receptor would foster a better understanding of the interaction between IL-2 and T-cells. To date this has been hampered, at least in part, by the unavailability of IL-2 receptor in purified form.
Leonard, et al, Nature 300: 267 (1982), reported employing a murine monoclonal antibody, designated anti-Tac, to significantly block the binding of radiolabeled IL-2 to the human lymphoma T-cell line, HUT-102. This antibody resulted from immunization of mice with long term cultures of human T-cells. The anti-Tac antibody was reported to bind to a glycoprotein receptor having a molecular weight of about 47,000-53,000 daltons, and also to proteins having molecular weights of about 113,000 and 180,000 daltons. Leonard, et al., suggested that the cell surface determinant (i.e., the 47,000-53,000 molecular weight protein) to which the anti-Tac antibody bound to was the IL-2 receptor.
Robb and Green, J. Exp. Med. 158: 1332 (1983), reported use of anti-Tac antibody in conjunction with mitogen-activated normal lymphocytes to immunoprecipitate a protein having a molecular weight of about 52,000-57,000 daltons. They found that this same protein also bound to IL-2.
Leonard, et al., Proc. Natl. Acad. Sci. USA 80: 6957 (1983), observed that receptors recognized by anti-Tac antibody on HUT-102 cells and on phytohemagglutinin-activated normal T-cells appeared to be larger on reducing gels than on nonreducing gels, thus suggesting the presence of intrachain disulfide bonds. Also, the HUT-102 cell receptor was reported to exhibit an isoelectric point of from 5.5 to 6.0. From post-translational studies, Leonard, et al., suggested that the HUT-102 receptor is composed of a peptide backbone of 33,000 molecular weight that is initially glycosylated by an N-linked mechanism to achieve a 35,000-37,000 molecular weight doublet and then glycosylated by an O-linked mechanism to increase the weight of the molecule by about 13,000-15,000 daltons.
The present invention provides a specific monoclonal antibody secreted by hybridoma 2A3 (ATCC HB 8555) that binds an epitope on the IL-2 receptor molecule with high affinity.
Preparation of IL-2 Receptor Extracts From Malignant and Normal Cells
Malignant cells are cultured in vitro in a suitable culture medium supplemented with serum and various additives. After an optimum culture period, the cells are harvested and IL-2 receptor containing extracts formed from the cells. The malignant cell lines which may be employed as a source of IL-2 receptors include T-lymphoma or T-leukemia cell lines. These cell lines are produced by either a spontaneous occurrence, via viral transformation or via transformation by chemical carcinogen or irradiation. The present invention has been carried out in conjunction with a naturally occurring lymphoma cell line, designated HUT-102. The cell line is available from a wide variety of sources and has been used extensively by researchers.
The numbers of IL-2 receptors expressed by stimulation of peripheral blood leukocytes with a plant mitogen varies with time. Optimum levels of IL-2 receptor expression are reached approximately 72 hours after mitogen stimulation.
The culture medium used to expand the IL-2 receptor bearing malignant cells may consist of commercially available medium, such as Roswell Park Memorial Institute medium (RPMI), Dulbecco's Modified Eagle Medium (DMEM) and Click's Medium. Additives, which may be individually or in combination added to the culture medium, include serum, such as fetal calf serum (FCS) or normal human serum. Additional additives include glutamine and various antibiotics, such as penicillin and streptomycin.
The process of culturing the malignant and normal cells to induce receptor formation may be carried out in various environmental conditions. Preferably, however, cultures are maintained in the temperature range of approximately 35�-38� C. in a humidified atmosphere of approximately 5-10% CO2 in air. Also, culture pH should be kept in slightly alkaline condition, in the range of approximately pH 7.0-7.4.
IL-2 receptor containing extracts are prepared from cultivated normal and malignant cells by harvesting the cells by centrifugation. The cells are then washed with a buffered saline solution and resuspended in the buffered saline solution together with a detergent and phenylmethylsulfonylfluoride (PMSF). After a period of time the detergent extract is centrifuged to remove nuclei and insoluble debris and then is stored frozen until used.
Preparation of Monoclonal Antibody Against IL-2 Receptor
The present invention provides a monoclonal antibody having a high affinity to an epitope on the IL-2 receptor molecule. The antibody is used as a bound ligand in the affinity chromatography procedures during purification of the IL-2 receptor. The antibody is also employed in a radioimmune precipitation assay and in soluble receptor assays to monitor the IL-2 receptor protein during purification steps, as more fully discussed below.
A preferred procedure for generating the monoclonal antibody against the IL-2 receptor is generally disclosed in U.S. Pat. No. 4,411,993, incorporated herein by reference. In the procedure, BALB/c mice are injected with PHA activated human peripheral blood leukocytes (PHA/PBL) several times at weekly intervals. Prior to the first injection, the PHA/PBL is emulsified in complete Freund's adjuvant and prior to the remainder of the injections the PHA/PBL is emulsified in incomplete Freund's adjuvant.
During the course of immunization, serum samples from the mice are tested by an enzyme linked immunoabsorbant assay (ELISA), as is well known in the art, for the presence of antibody reaction with the immunization cells. Once an antibody titer is detected, the animals are given an intravenous injectoion of PHA/PBL suspended in saline. Several days later the animals are sacrificed and their spleens harvested. Single cell suspensions from the splenocytes are cultured in tissue medium supplemented with various additives to expand the number of antibody producing cells. The antibody producing cells are isolated from the culture and purified by standard techniques for subsequent fusion with myeloma cells to produce hybrid cells that express anti-IL-2 receptor antibody. The fusion process is detailed in U.S. Pat. No. 4,411,933 and in Nowinski, et al., Virology 93: 111 (1979), incorporated herein by reference.
After fusion, the hybrid cells are resuspended in a tissue culture medium supplemented with various additives and selected suppressing agents to preclude the growth of unfused myeloma cells, double myeloma cells, unfused spleen cells and double spleen cell hybrids, thereby liberating the anti-IL-2 receptor antibody producing cells. Such growth inhibitors or suppressants may include hypoxanthine, aminopterin and thymidine (hereinafter collectively referred to as HAT).
After several days of culture, the hybridoma cells which are generated are screened by ELISA assay for anti-IL-2 receptor antibody responses. These hybrid cells are tested for production of antibody capable of inhibiting both mitogen and antigen induced proliferation of human peripheral blood leukocytes. The hybrid cells which give positive ELISA results are gradually weaned to HAT-free medium and then cultured in vitro in large volumes for bulk production of antibody. Alternatively, the cells may be expanded in vivo by injecting the hybridoma cells in the peritoneal cavities of mice and thereafter collecting the intraperitoneal ascites which contain high concentrations of the antibody. The antibodies contained in the ascites fluid can be isolated and concentrated by established techniques, such as by ammonium sulfate precipitation followed by gel column chromatography. If required, the antibody can be further purified by ion exchange chromatography and/or affinity chromatography. By the above process, a particular hybridoma, designated as 2A3, was found to produce antibody that significantly inhibited both mitogen and antigen induced proliferation of human peripheral blood leukocytes.
The present invention concerns potent cell line sources of anti-IL-2 receptor antibody by cloning cell lines known to produce this antibody, for instance, the 2A3 cell line. The cloning is accomplished by the limiting dilution procedure, as is well known in the art and as is detailed in U.S. Pat. No. 4,411,993. By this procedure, one particular subclone, designated 2A3-A1H, was found to produce antibody that almost entirely inhibited both mitogen and antigen induced proliferation of human peripheral blood leukocytes. The 2A3-A1H antibody has been characterized as of the γ1 K isotype with an unusually high affinity to the human IL-2 receptor. The 2A3 hybridoma cell line has been deposited with the American Type Culture Collection, Rockville, Md., USA, (ATCC) under accession number ATCC HB 8555.
A control antibody preferably is employed to confirm the processes of the present invention utilizing anti-IL-2 receptor antibody and as a reagent in the purification of the receptor. The control antibody should be of the same isotype as the anti-IL-2 receptor antibody. Applicants have identified the antibody secreted by the mouse myeloma cell line MOPC-21 as a suitable control antibody for the 2A3-A1H antibody. The MOPC-21 cell line is widely available from numerous private and commercial sources.
Soluble IL-2 Receptor Assays
Assays employing the 2A3-A1H monoclonal antibody are used in conjunction with the present invention to monitor the quantitative amount of IL-2 receptor present in the initial cell lysates and during purification procedures. These assays hinge on the discovery by applicants that the 2A3-A1H antibody has an extremely high affinity for the IL-2 receptor, the affinity constant being in excess of 5�109 per M-1 and that the 2A3-A1H antibody can be radioiodinated to high specific activity and still retain its capacity to bind to the IL-2 receptor.
One such preferred assay involves ascertaining the extent to which samples of cell lysate or column chromatography fractions containing IL-2 receptors are capable of inhibiting the binding of radiolabeled IL-2 antibody to glutaraldehyde fixed, intact receptor bearing cells. This assay relies on the observation by applicants that IL-2 receptor is stable to glutaraldehyde fixation, i.e., the receptor cannot be extracted from such cells with nonionic detergents, such as Triton X-100, and the presence of detergent does not affect the binding of radiolabeled 2A3-A1H antibody to the fixed cells. Preincubation of a subsaturating dose of iodinated 2A3-A1H antibody with detergent solutions containing the IL-2 receptor inhibits the subsequent binding of the 2A3-A1H antibody to the glutaraldehyde fixed cells. This assay will hereinafter be referred to as the soluble inhibition assay.
For use in the soluble inhibition assay, the 2A3-A1H antibody is radiolabeled with iodine 125 (125 I) by a chloramine-T method, as is well known in the art and as described by Segal and Hurwitz, J. Immunol. 118: 1338 (1977). The standard labeling conditions employed are: 50 μg 2A3-A1H IgG; 4 nmoles of chloramine-T (Sigma Chemical Company, St. Louis, MO); and, 2.5 mCi of 125 I sodium iodide (New England Nuclear, Boston, MA), in a final volume of 60 μl. This protocol has resulted in preparations of 125 I-2A3-A1H, which routinely have specific activities in the range of 2 to 5�1015 cpm/mmole (1.3-3.3�107 cpm/μg). Also, 2A3-A1H antibodies labeled in this way were found to be more than 95 percent bindable to IL-2 receptor bearing cells and had apparent affinity constants in excess of 5�109 M- 1.
In the soluble inhibition assay, 50 μl of 125 I-2A3-A1H (2�10-10 M) is mixed with 50 μl of an appropriate dilution of cell lysate or column fraction in phosphate buffered saline (PBS) containing 1% (w/v) Triton X-100 detergent (Sigma Chemical Company, St. Louis, MO) in RPMI-1640 medium containing 2% bovine serum albumin (BSA), 20 mM HEPES buffer (pH 2.7) and 0.2% sodium azide (binding medium). This mixture is incubated for one hour at room temperature in round bottom 96 well plates (Linbro, Hamden, CT). At the end of the incubation period, 107 glutaraldehyde fixed, PHA activated human T-cells in 50 μl of binding medium are added to detect uncomplexed 125 I-2A3-A1H. Incubation is continued for one hour at room temperature. Duplicate 60 μl aliquots of the mixture are then transferred to precooled 400 μl polyethylene centrifuge tubes containing 200 μl of a phthalate oil mixture and the cell bound antibody is separated from unbound antibody by centrifugation. The details of the well-known phthalate oil separation method are set forth in Segal and Hurwitz, supra. The percent of specific inhibition caused by the lysate or column fraction is calculated by using 50 μl of PBS-2% Triton X-100 instead of a test sample for the positive control. Also, 15 μl of PBS-2% Triton X-100 containing 10-8 M unlabeled 2A3-A1H is used as a negative control.
The nitrocellulose dot assay (dot assay) is used as a second soluble IL-2 receptor assay to quantify the amount of IL-2 receptor molecules present in a sample of cell lysate or column fraction. Briefly, in the dot assay, solutions are made of a log2 dilution series of potential IL-2 receptor containing samples and PBS containing 1% Triton-100. Samples of 5 μl of these solutions are then applied to a piece of dry nitrocellulose (Schleicher and Schvell, Keene, N.H.). The nitrocellulose is incubated for one hour at room temperature in 10 ml of TBS-3% BSA containing 0.05 μg/ml 125 I-2A3-A1H and 0.6 μg/ml unlabeled MOPC-21. The nitrocellulose is then washed three times in TRIS buffered saline and twice in TRIS buffered saline containing 1% (w/v) Nonidet P-40 detergent (Gollaro Schlesinger Chemical Manufacturing Corp., Carl Place, N.Y.), 1% (w/v) sodium deoxycholate, and 0.1% (w/v) sodium lauryl sulfate. Each of these washes lasts 30 minutes at room temperature. After the final wash, the nitrocellulose sheet is blotted dry, covered with a clear plastic sheet and then exposed at -70� C. to Kodak X-omat AR film.
Radioimmune Precipitation Assay
The specificity of the IL-2 receptor antibody is ascertained with a radioimmune precipitation assay involving forming precipitations between samples of radiolabeled IL-2 receptor molecules and an antibody to the receptor and then employing polyacrylamide gel electrophoresis and either fluorography or autoradiography to visualize the receptor proteins that were precipitated. In this assay technique, the IL-2 receptor molecules are labeled either by surface iodination after extraction or metabolically before extraction.
Radiolabeling of the IL-2 receptor cell membranes after extraction is performed by the 125 I-IODO-GEN method (Pierce Chemical, Rockford, IL). The details of this radiolabeling technique are well known in the art and described by Urdal, et al., Cancer Metastasis Reviews 1: 65 (1982); and, Markwell, et al., Biochemistry 17: 4807 (1978). The use of 35 S methionine to label the receptor molecules metabolically also is well known in the art and is described by, for instance, Robb and Greene, J. Exp. Med. 58: 1332 (1983).
After labeling with 125 I or 35 S methionine, the cells are washed with PBS and then extracted with PBS containing 1% Triton X-100 and 2 mM PMSF. Affinity supports for the radioimmune precipitation assay are prepared by coupling purified antibodies (2A3-A1H and MOPC-21) to Affi-gel-10. Briefly, one volume of moist Affi-gel-10 is added to one volume of antibody (3-5 mg/ml) in borate buffered saline (BBS) and then the mixture incubated overnight at 4� C. Thereafter, 100 μl of 1M glycine ethylester is added per ml of gel to couple any of the unreacted groups on the Affi-gel-10. Applicants have found that routinely from 3 to 4 mg of antibody are coupled per ml of the gel. Before use, each gel is washed extensively with PBS. Each gel is also washed with a buffer solution composed of PBS-1% Triton X-100 and 0.5M TRIS, pH 7.5, containing 0.5M NaCl, 1% (w/v) NP 40 detergent, 1% (w/v) sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) (RIPA buffer).
The radioimmune precipitations are performed by mixing 50 μl of radiolabeled cell extract with 75 μl of PBS-1% Triton X-100 containing 20% (v/v) of affinity gel having antibody coupled thereto. The mixture is incubated overnight at 4� C. and then the gel washed four times with RIPA buffer and twice with 0.1M TRIS, pH 8.0, containing 0.5M NaCl, 5 mM ethylenediamine tetraacetate (EDTA), and 0.5% NP-40 detergent. After the final wash, the resultint gel pellets are suspended in 40 μl of SDS polyacrylamide gel sample buffer (0.06M TRIS, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and boiled for three minutes to break apart the bonds between the antibody and the IL-2 receptor molecules. A 30 μl sample of the supernate is then analyzed by SDS-Polyacrylamide gel electrophoresis (PAGE) (8% polyacrylamide gel for 125 I labeled receptor; 12% polyacrylamide gel for 35 S methionine labeled receptor) according to the stacking gel procedure of Laemmli, Nature 227: 680 (1970). Receptor proteins are visualized on gels by autoradiography.
Gel Electrophoresis of Chromatography Column Fractions
Fractions eluted from the affinity chromatography and reversed phase HPLC columns employed in the purification processes of the present invention are assayed by gel electrophoresis. 50 μl aliquots were removed from the eluate fractions. The aliquots are dried under vacuum after addition of 2 μl of 10% SDS (w/v) to each aliquot. The dried residue is dissolved in 40 μl of SDS polyacrylamide gel sample buffer and then boiled for 3 minutes. The solution is applied to an 8% polyacrylamide gel and electrophoresis is then carried out by the stacking gel procedure of Laemmli, supra. The resulting gel samples are silver stained by the method described by Oakley, et al., Anal. Biochem. 105: 361 (1980).
Purification of IL-2 Receptor
Cell extracts from the malignant and normal cells produced by the above procedures are initially concentrated by affinity chromatography techniques employing the same affinity supports used in the radioimmune precipitation assay described above. The procedure employed involves applying cell extracts first to an MOPC-21 column and then to a second column prepared from a mixture of MOPC-21 antibody and 2A3-A1H antibody so that in the second column from 3 to 4 mg of total IgG is coupled to each ml of gel, but only 10 to 30% of the antibody is composed of 2A3-A1H. This technique is used to counteract the extremely high affinity between the 2A3-A1H antibody and the IL-2 receptor.
In the purification procedure, the cell extracts, as prepared above, are first applied to the MOPC-21 column that has been preequilibrated with an appropriate buffer containing a detergent, thereby to remove proteins in the cell extract that might nonspecifically bind to mouse immunoglobulin. The flow through from the MOPC-21 column is then applied to the 2A3-A1H column. Elution is carried out with an appropriate saline-detergent solution. The recovered fractions are then dialyzed against decreasing concentrations of the eluting agent to optimize the recovery of biological activity.
Fractions are collected and assayed by gel electrophoresis and silver staining, as described above. Applicants have found that by use of the affinity chromatography procedure, IL-2 receptor from malignant cells which constitutively produce the receptor is purified approximately 1600 times from initial cell lysate. A somewhat lower purification level is typically attained for IL-2 receptor from activated normal cells.
The pooled active fractions from the above affinity chromatography procedure is employed as a starting material for the HPLC procedures. The HPLC technique used in the present invention preferably employs a reversed phase, tetra methyl bonded silica column having a pore size sufficiently large to be optimally utilized with the proteinaceous IL-2 receptor, i.e., a pore size of at least 300 A.
Suitable reversed phase HPLC columns for use in the practice of the present invention are articles of commerce. A preferred column for this purpose is the Vydac C-4 reversed phase column commercially available from Separations Group, Hesperia, CA. This column consists of tetra methyl silane groups covalently bonded by means of a siloxane (silicon-oxygen-silicon) bond to the surface of the 300 A pore diameter silica gel which has been classified to a mean particle size of 5 microns.
The elution of the proteins from the HPLC column is carried out in a manner well-known in the art. A suitable elution procedure for removing the bonded receptor molecule proteins from the tetra methyl column involves the use of a linear gradient of the acetonitrile. A preferred gradient for this purpose is 0 to 95% (v/v) acetonitrile gradient in trifluoroacetic acid (TFA), pH 2.1.
The eluted protein can be conveniently monitored with detection systems that are well-known in the art. For example, an automated fluorescence detection system as described by Stein and Moschera, Methods Enzymol. 78: 435 (1981), may be employed. Alternatively, the relative protein concentration in the fractions eluted from the HPLC columns can be determined by measuring absorbance of the eluted material in an automated ultraviolet light spectrophotometer, at 214 nanometers light wave length. The suitable automated ultraviolet light absorbance detection apparatus is available from Waters Associates, Milford, MA.
By use of the above described soluble receptor assay techniques, applicants have found that the specific activity of the IL-2 receptor after HPLC purification is very high, i.e., approximately 21,000 femtomole (fm) IL-2 receptor/μg protein for IL-2 receptor derived from malignant cells. This is approximately a 16,700 fold level of purification over the specific activity of the IL-2 receptor in the starting cell lysate. The specific activity of the IL-2 receptor from normal T-cells was about 1/3 of the specific activity from malignant cells. By polyacrylamide gel electrophoresis and silver staining, applicants ascertained that the molecular weight of the IL-2 receptor from normal cells is approximately 60,000 daltons, as opposed to 55,000 daltons for receptor molecules found constitutively on the malignant cells.
Amino Acid Sequencing
The ability to prepare homogeneous IL-2 receptor has permitted applicants to determine the amino acid composition and sequence of the amino terminal portion of this molecule. This information may be employed to assist in the cloning of the IL-2 receptor gene and the production of large quantities of pure IL-2 receptor for clinical trials and ultimately for widespread medical use. Moreover, the availability of homogeneous IL-2 receptor will no doubt lead to a more complete understanding of the biology of IL-2.
Samples of homogeneous IL-2 receptor, as prepared above, can be analyzed for amino acid composition and sequence, for instance with an automated sequencer, such as with an Applied Biosystems model 470A protein sequencer. Ideally, several sequencing runs are made to confirm the accuracy of the sequence. Through this technique, applicants have found that the first 15 residues of the amino terminal portion of the IL-2 receptor molecules are composed of the following sequence: Glu-Leu-Cys-Asp-Asp-Asp-Pro-Pro-Glu-Ile-Pro-His-Ala-Thr-Phe.
The processes and products of the present invention are further illustrated by the following examples.
Preparation of IL-2 Receptor-Containing Extracts From Malignant Cell Line
HUT-102 cells in a concentration of 2�105 cells per ml are cultured in 100-500 ml volumes in various plastic culture flasks or bottles (Falcon Plastics, Oxnard, CA) in RPMI-1640 medium. The medium is supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Since the HUT-102 cells have been reported to produce human T-cell leukemia virus (HTLV-1), work with this cell line is performed in a P-3 isolation facility.
The cells are cultured for 3-5 days in a humidified atmosphere of 5% CO2 in air. After this period of time, viable cells are harvested by centrifugation and washed three times in PBS. Thereafter, the cell pellet is suspended in a volume that is three times the volume of the cell pellet in a solution composed of PBS containing 1% (w/v) Triton X-100 detergent and 2 Mm PMSF. This mixture is kept on ice and periodically vortexed for 30 minutes. The extract was then centrifuged at 20,000�g for 20 minutes to remove nuclei and insoluble debris. The cell extract, as thus prepared, is then stored at -70� C. until used.
Preparation of IL-2 Receptor-Containing extracts From Lectin Activated Normal Cells
Human peripheral blood mononuclear cells are prepared by Ficoll-Hypaque density gradient centrifugation as described by Boyum, supra. Briefly, enriched T-cells are obtained by rosetting with neuraminidase-treated sheep erythrocytes. The resulting rosetting (E+ () and nonrosetting (E-) cells are incubated separately in 100-mm plastic petri dishes in 8% FCS at a concentration of 2-5�106 per ml. The adherent E- cells are recovered with a rubber policeman after removing nonadherent cells with three media washes. The E- adherent cells together with the E+ nonadherent cells in a ratio of 1:25 are placed in bulk culture in 75-cm2 flasks at a concentration of about 1-2�106 cells/ml in RPMI-1640 medium supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. Activation is accomplished with 1% (v/v) PHA (Difco Laboratories, Detroit, MI). The cultures are incubated at 37� C. in a humidified atmosphere of 5% CO2 in air. Aliquots containing approximately 1-2�107 cells are removed at various times for analysis of cell surface IL-2 receptors.
Cells are harvested by centrifugation approximately 72 hours after mitogen stimulation, and washed three times with PBS. The resulting cell pellet is suspended in a volume three times the volume of the pellet in a solution composed of PBS containing 1% (w/v) Triton X-100 detergent and 2 mM PMSF. The resulting mixture is kept on ice with periodic vortexing for 30 minutes. Thereafter, the extract is centrifuged at 20,000�g for 20 minutes to remove nuclei and insoluble debris. The resulting cell extracts are stored at -70� C. until used.
Production of Monoclonal Antibody to IL-2 Receptor
Female BALB/c (Jackson Laboratories, Bar Harbor, ME) of ages from 8-12 weeks are immunized intradermally in the hind legs with 107 PHA/PBL cells. Prior to immunization, the PHA/PBL cells are prepared as an emulsion by mixing these cells with 0.4 ml of complete Freund's adjuvant (Difco Laboratories, Detroit, MI). After the initial immunization, the mice are rechallenged weekly for four weeks with 107 PHA/PBL cells in incomplete Freund's adjuvant.
Periodically, serum from the mice is collected and tested individually for binding to PHA/PBL by ELISA, in a manner well known in the art. The animals found to hve the highest response are given an additional intravenous injection of 107 PHA/PBL in PBS. Four days later, the animals are sacrificed by cervical dislocation. The spleens of the animals are harvested and single cell suspensions prepared therefrom. The spleen cells are cultured in medium.
Fusion is achieved by mixing approximately 20�106 spleen cells with approximately 10�106 NS-1 murine myeloma cells in a 50 ml conical centrifuge tube. The cell mixture is pelleted by centrifugation for 5 minutes at 200�g, and then the supernate removed by aspiration. The centrifuge tube with its intact cell pellet is transferred into a 37� C. water bath. Then polyethylene glycol 15 w (Eastman, Inc.) 50% (w/v) in RPMI-1640 is added to the cell pellet in dropwise manner at a ratio of 1 ml of PEG/1.6�108 spleen cells. Thereafter, one volume of RPMI-1640 and 10 volumes RPMI 1640 containing 15% FCS and 1 mM pyruvate are slowly added during gentle stirring. Then, the cell suspension is centrifuged at 200�g for 5 minutes and the supernate discarded to complete the fusion process.
The hybrid cells are selected by resuspending the resulting cell pellet in Click's medium containing 15% FCS and 100 mM sodium pyrate. The unfused myeloma driver cells (NS-2), double NS-1 hybrids, unfused spleen cells and double spleen cell hybrids are prevented from proliferation by the addition to the medium of approximately 13.6 mg/L of hypoxanthene, 0.176 mg/l aminopterin and 3.88 mg/L of thymidine. The suspension is then divided into 200 μl aliquots in flat-bottom microtiter plates (No. 3596, Costar Inc., Cambridge, MA). The cultures are maintained at approximately 37� C. in a humidified atmosphere of 5% CO2 in air.
After 10 days of culture, a 100 μl aliquot of supernate is removed from each viable culture and tested in an ELISA assay for binding to PHA/PBL (IL-2 receptor positive) or PBL (IL-2 receptor negative). Hybrids which demonstrate significant binding to PHA/PBL and little or no binding to PBL are transferred to 1 ml cultures and gradually weaned to HAT-free media. These hybrids are subcloned by limiting dilution cultures. Through this process, applicants have identified one particular hybrid clone, designated as 2A3-A1H, which significantly inhibits both mitogen and antigen induced proliferation of human PBL. The 2A3-A1H monoclonal antibody has been characterized as of the γ1 K isotype that exhibits a very high affinity to the human IL-2 receptor. This antibody inhibits the binding of IL-2 to its receptor and is antagonistic of IL-2 action.
In Vivo Production of Hybridoma Cells Producing Monoclonal Anti-IL-2 Receptor Antibodies
Anti-IL-2 receptor antibody was produced in high concentration in vivo by intraperitoneal injection of BALB/c mice with approximately 1-10�106 hybridoma cells. One week prior to hybridoma cell injection, recipient BALB/c mice were given approximately 1.0 ml of pristane intraperitoneally as an ascites inducing irritant. From 8 to 14 days after hybridoma injection, intraperitoneal ascites were collected and each volume of fluid is mixed with 0.9 volume of 45% saturated ammonium sulfate and stirred overnight. The precipitate is separated by centrifugation and redissolved in phosphate buffer (0.05M), pH 6.8. Residual ammonium sulfate is removed by dialysis against the same buffer.
The protein solution is then passed over a 5 ml bed volume DE-52 column (Whatman, Clifton, NJ) and the fronting peak of protein is pooled. The pooled fractions are dialyzed against 0.02M sodium borate, 0.1M sodium NaCl, pH 8.5, (BBS) and then applied to a 2.6�90 cm ACA-34 (LKB, Bromma, Sweden) gel filtration column previously equilibrated in the same buffer. The fractions corresponding to IgG are collected and pooled. Yields typically are in the range of 3 mg IgG/ml of ascites.
Purification of IL-2 Receptor by Affinity Chromatography
Cell extracts from normal and malignant cells produced by the procedures of Examples 2 and 3 are concentrated by affinity chromatography technique employing an initial gel column having control antibody for removing protein that might nonspecifically bind to mouse IgG and a second column having 2A3-A1H antibody bound thereto. The control antibody used in the initial column is secreted by the myeloma cell line MOPC-21. This antibody is of the same isotype as the 2A3-A1H antibody and is readily available.
To prepare the columns, purified 2A-3-A1H and MOPC-21 antibodies are coupled to Affi-gel-10 (Biorad, Richmond, CA) according to the manufacturer's instructions. Equal volumes of moist Affi-gel-10 and antibody (3-5 mg/ml) in PBS are mixed together and incubated overnight at 4� C. Thereafter, unreacted sites on the Affi-gel-10 are blocked by addition of 100 μl of 1M glycine ethyl ester per ml of gel. Applicants found that the antibody-coupled gel routinely contained from 3 to 4 mg of antibody per ml of gel.
Because the 2A3-A1H antibody exhibits such an extremely high affinity for the IL-2 receptor, the receptor yield from the chromatography columns was improved by employing columns prepared with a mixture of MOPC-21 and 2A3-A1H antibody. A total of 3 to 4 mg IgG is coupled per ml of gel, of which only 10-30% of bound IgG is 2A3-A1H.
Prior to use, each gel is washed extensively with PBS and RIPA buffer. The MOPC-21 and 2A3-A1H gel columns are poured in 3 ml syringes that have their open ends closed with a cork and tubing, thereby to enable the columns to be run in either direction. The cell extracts, as prepared in Examples 1 and 2 above, are first applied to the MOPC-21 column at a flow rate of 0.1 ml/min at 4� C. to remove proteins that nonspecifically bind to the mouse IgG. This absorption is repeated once more and then the flow-through from the MOPC-21 column is twice applied to the 2A3-A1H column.
The 2A3-A1H column is then washed with 10 column volumes of PBS-1% Triton X-100, 10 column volumes of RIPA buffer and lastly, 10 column volumes of PBS-1% Triton X-100. Thereafter, the receptor is eluted from the column with 6M guanidine hydrochloride (GuHCl) and 0.5% Triton X-b 100. Eluate fractions in 1.2 ml volume are collected and each fraction is dialyzed against 3M GuHCl in 0.5% Triton X-100 for four hours. This is followed by dialysis against 1.5M GuHCl in 0.5% Triton X-100. Final dialysis is performed against PBS containing 0.5% Triton X-100. Aliquots at each stage of the purification are saved for analysis of biological activity by the above-described soluble receptor assays; protein concentration by fluorescamine assay with bovine serum albumin as a standard, as is well-known in the art; and, protein heterogeneity by polyacrylamide gel electrophoresis with the protein being detected by silver staining, as also described above. From these assays, the IL-2 receptor from the HUT-102 cells was found to have a specific activity of approximately 2,000 fm receptor/μg protein. The specific activity from the PHA/PBL cells was somewhat less.
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|International Classification
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|Dec 17, 1986
Owner name: IMMUNEX CORPORATION, 51 UNIVERSITY STREET, SEATTLE
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|Jan 23, 2001
|Maintenance fee reminder mailed
|Jul 1, 2001
|Lapse for failure to pay maintenance fees
|Sep 4, 2001
|Expired due to failure to pay maintenance fee
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Megan from Brassy Apple is here to share another great idea for Funner in the Summer: a Summer Food List. What a simple but brilliant idea. She just announced her new show, Refashion It, on My Craft Channel- congrats Megan!!Hey! It's Megan of Brassy Apple! It's fun and great to have Summer Bucket lists and along those lines we created a Summer FOOD list! There … [Read more...]
Pity Party 56
If you are new to 30days, I host a linky party, my Pity Party to link up to every Friday. I love checking out new links and finding new blogs to visit.Image via Epiphanie bags The Rules:Link to a specific post, not your blog in general. No links to shops, products or giveaways please. (They will be deleted) Be creative- a craft, tutorial, recipe, anything you … [Read more...]


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2 volumes
For many businesses, compliance with environmental protection legislation is now just as important as compliance with the Companies Acts. Equally important is full regard for environmental liability when entering into everyday commercial and commercial property transactions. Commercial Environmental Law and Liability is the commercial and commercial property lawyers' guide to environmental protection legislation; the procedures to follow; the insurance cover available; and the environmental compliance requirements for the development of business operations. This is supported by the necessary background information on environmental protection legislation, including the general principles, measurement and controls, regulatory bodies, the EU and enforcement of the law. There are also forms and precedents for entering into transactions and seeking permission to undertake new operations. * Examines commercial environmental liability in relation to everyday issues faced by commercial law practitioners * Looks at compliance procedures for efficient administration * Sets out the safeguards for commercial and property transactions so that environmental liabilities can be identified and dealt with * Contains forms and precedents for commercial and property transactions * Details the procedures to follow when developing business operations which will be affected by environmental legislation
Professional advisors whose areas of practice are restricted to commercial and property law will find it exceptionally usefulCertainly no firm which practices commercial law or commercial property law should be without it. Estates Gazette.
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This gorgeous hotel has recently undergone a multi-million dollar renovation. The luxurious property offers casual elegance in a stunning atmosphere. It sits directly on one of the most beautiful beaches in the world and there is plenty to do on-site for couples and families alike. The hotel has 159 rooms spread over 12 floors. It is air-conditioned and has a 24-hour check-in service, hotel safe, cloakroom, lift access, kiosk, 2 bars, 2 restaurants, room service, complimentary internet access and a laundry service (available weekdays only). Parking facilities are available.
This charming property is located along the Palm Beach coastline in Jupiter. The hotel sits on the largest stretch of private beach in Palm Beach County. Guests will be just minutes from world-class shopping, dining, entertainment, and golf. In addition, the hotel is only about 28 km north of Palm Beach International Airport.
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Gem 9/27/13 Sailing: Norwegian Gem Cruise Review by Jst4fun137
Overall Member Rating
Gem 9/27/13 Sailing
Destination: Canada & New England
Embarkation: New York (Manhattan)
Our first port was Halifax, we did an excursion through the ship. Peggy's Cove and the Titanic Cemetary. It was just the two of us so it was cheaper and easier to go through the ship. In canada they like to charge taxis by the hour so if you have a group that would work better for you.
In St. John's we did the jet boat ride at the reverse falls. I booked it through them directly and it was much cheaper with a 10 min taxi ride of 10.00. The ride was amazing, you do get wet. We ended up not using our change of clothes and More just took a taxi back to the ship wet.
In Bar Harbor, I booked Olie's Trolley on the internet so we didn't have to wait it line. Arcadia was closed, but the tour company did a great job in making changes to the itineary. I would recommend The Thirsty Whale for a great bowl of chowder and Lobster Roll at a great price. CJ's ice cream had great Blueberry Ice Cream.
In Boston, I also went on line ahead of time for the Hop on Hope off Orange and Green Old Town Trolley, you get an online discount. We did the free water cruise that came with the ticket. We opted to do that first to get it out of the way. Saw Paul Revere's house and went to Mike's Pastery. The only problem was once we were on the Trolley, we were afraid to get off at certain stops due to the large amount of people waiting to get on. We were afraid we wouldn't get back to the ship in time. All the drivers were very nice.
In Newport, I had gone on The Historical Society website and did the Elms Tour with the Servant Life tour. We did the Elms first and the Servant Life tour was at that house as well. It also included a lunch and we had a sandwich before heading to the Breakers. Its a good walk between the too. We did the Breakers but due to the time, we didnt do Marble House which was included in my price all for 49.00. We walked around where the ship tendered in, so if we hadn't done that we would have made it all. All in all we had a great time.
We stayed on the ship Saturday morning until 9:45 and just walked right off. There wasnt a very long line at Customs so we breezed right through. Walked right across the street for a taxi to LGA. We had been assigned a handicap stateroom (not sure why) so there was a little more space then normal Just had to get used to the handles and buttons. Our cabin steward was great. Ask for pool towels to take to the jet boat ride and he got them for us. All the staff and crew went out of their way in any way they could. Dan Dan the cruise director was funny. The production numbers were ok. The dancers were great, but they only had comedians on board so didnt do the shows every night. Have liked other NCL ship shows that had more different entertainment. Lounge acts werent as exciting as on other NCL ships. There were quite a few handicapped folks. Lots of walkers, chairs and just plain old slow people. A lot of the passengers were very rude, especially at the buffet. People acted like they've never eaten before and that they were never gonna be able to eat again. I would have to say that was the worst part of the whole trip was the passengers attitudes. Less
Published 10/19/13
Cabin review:
Great location, right off elevator. Was a handicap room, so there was a button to open/close deck door. There were ramps at bathroom and deck door. The bathroom just had a shower curtain with drains in the floor. Nothing that was difficult to adjust to.
Port and Shore Excursions
City Tour
Arcadia was closed due to the shut down. They did a decent job of filling in, since they didnt have a lot of time or other things to choose from
Old Town Trolley Tour
You get a discount if you book online. They are right at the end of the cruise pier under a white tent. You can purchase tickets directly from them. Not a lot of trolleys running for the amount of people riding. Missed some things we wanted to see because we were afraid to get off for fear of not getting back in time. Stops at the front of the line were good. It was the stops further out like Fenway and Back Bay that were the problem
Peggy's Cove
Tour guide was good. Sit on the side behind the driver to see all the stuff the driver talks about. Had gingerbread at shop at Peggy's Cove It was good Also went to Titanic Cemetary
Breakers Mansion
crowded but interesting. All tours are self guided
Cliff Walk
3 homes, The Elms. Breakers and Marble House with the servant life tour and lunch for 49.00 The cliff walk you can do on your own
The Elms Mansion
was very interesting to see how these people lived
Jet Boat
Awesome ride. Highly recommended. Very friendly staff
Read Cruise Critic's Norwegian Gem Review >>
An overall great cruise
Exceeded expectations
Great itinerary, so so food
Love the GEM
Frat Party Central
Overall very good first experi...
Not the best, but definitely n...
Great time but it was the comp...
Third time on the Escape. Firs...
Sponsored Links


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Product Description
Style your hair, and if you change your mind, restyle it with this Style Sexy Hair Molding Paste. The pliable formula allows you to manipulate hair until you get the style that you want, and its semi-matte finish won't look greasy. Sculpt and create a hair masterpiece with awesome staying power.
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Companies involved in unethical business practices don't stay in business very long. Today's consumers are savvy and do not want to do business with a company lacking strong morals and ethics. The Garland Company is happy to be a company that has stood the test of time. We pride ourselves on being successful in business for over a century, thanks in large part to our stringent ethical policies.
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They don't prosecute or fine you for failing to respond, but since I'm a pollster, I respond for fun. It's entertaining to see how "truthful but not helpful" a response can be written. For example, I don't have running water because none of the taps were on when I answered. I'd rather mess with them than ignore them, but either option is worthwhile.
Running water?
I bet FDR put that question in there.


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25 Table Setting Ideas to Inspire Your Next Party
Find fresh, pretty table setting ideas for any occasion, and create the look yourself with easy tips and trends.
You Know Hue
The vibrant yellow pattern of this china is the focal point of this outdoor table. All-white linens really help the pattern shine, and pink-and-yellow centerpieces repeat the colors of the china.
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Salaam Remi - One in the Chamber ft. Akon MP3
From the Grammy nominated album One: In The Chamber, available now at iTunes http://smarturl.it/salaamremichamberdig Music video by Salaam Remi feat.
Salaam Remi feat. Akon - One in the Chamber (Lyric) MP3
This is a lyric of a new song from Akon feat. Salaam Remi.
Salaam Remi - One in the Chamber ft. Akon MP3
one chamber -uploaded in HD at http://www.TunesToTube.com.
[Original Song] One In the Chamber - XTREAMEGaming31 (Jayson J) MP3
This is my first Drum ans Bass song. Enjoy! All right reserved. Tags; Music EDM Drum and Bass FL Studio Song Original.
Akon - So Blue MP3
Download "So Blue" now on: iTunes: http://smarturl.it/AkonSoBlueiT Amazon: http://smarturl.it/AkonSoBlueAZ Google Play: http://goo.gl/Szz4Ro Music video by ...
So Blue
AkonI see you every damn day and I can't help wondering why Why you look so good, but I ain't ever seen you smile Got a man that defined you, everything in this world But I guess he ain't loving you right, girl, no Cause I, I can see that you're lonely Baby, you're one and only He ain't giv[...]
Akon - Each His Own (WORLD PREMIERE live at TRACE Urban Music Awards 2014) MP3
Watch Akon performing his new single Each His Own live at TRACE Urban Music Awards 2014 in Paris! This song is from his upcoming album Stadium which is ...
* * *Honorebel ft Akon - One More Slam (NEW!!! 2010!!!)* * * MP3
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OMI feat. Nicky Jam - Cheerleader (Felix Jaehn Remix) [Cover Art] MP3
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Headstones - the making of One In The Chamber Music MP3
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Each His Own Each His Own Each His Own Each His Own Each His Own Each His Own Each His Own.
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Akon - So Blue (lyrics) MP3
So Blue song by Akon, lyric video edited by Leonardo Matheus This is the new single from STADIUM album coming soon in the end of year So Blue new song ...
Beres Hammond - One Love, One Life (album mix) MP3
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Akon - Rely On (Official Audio) MP3
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REVIEW SNAPSHOT®by PowerReviews
Reviewed by 1 customer
Comments about Spencer's Dual Tower Speakers:
These speakers are definitely worth every penny and more. Personally I am shocked at how clean the sound is, very very neutral sound. There is no bass to these puppies at all which is my only con. But you can't expect such clean sound and bass at the same time. These are as crisp and clean sounding as my studio monitors (MDR V6's). Very very impressed.


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Your Chance at the 99-point "Italian Wine of the Year" for Just $29.99
Your Chance at the 99-point "Italian Wine of the Year" for Just $29.99
Posted February 25, 2015
WineLibrary Staff
THIS WINE IS SOLD OUT! Thanks to everybody who grabbed a bottle.
2009 Nativ Aglianico 'Eremo San Quirico' Taurasi
99 Points - Luca Maroni
List Price - $50.00
WL Sale Price - $29.99 (40% Off!)
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We love because he first loved us.
1 John 4:19
Written by Tapestry Treasures
1 John 4:19
Soldier's Duty "Thou therefore endure hardness, as a good soldier of Jesus Christ. No man that warreth entangleth himself with the affairs of this life; that he may please him who hath chosen him to be a soldier." (2 Timothy 2:3-4) As Paul came to the end of his earthly life, he took great pains […]
Originally posted on THE RIVER WALK:
Give me the wisdom and knowledge to lead them properly, for who could possibly govern this great people of yours? (2 Chronicles 1:10) Read: 2 Chronicles 1:1-3:17, Romans 6:1-23, Psalm 16:1-11, Proverbs 19:20-21 Relate: Last night I was talking with someone who had just left a board meeting with a…
It's time to seek the Lord!
When he comes, he will rain righteousness on you.
"God is breaking new ground in me. He is helping me cultivate my heart to receive His plans and His purposes. As I seek Him, I am growing. It is not me waiting upon the Lord, but rather, the Lord waiting upon me. I must grow into His purpose. I must grow into His call. His good plans for me are so beautiful and great that I could never fulfill them in my seed form. So, I say, Lord be it unto me according to your purpose. I give up my seed and allow your transformation. I accept this dark time as my place to germinate in your Holy Spirit. I die to my world(my way of doing and thinking) even as you are rooting me and drawing me forth. I know what you are doing is great! I know that spring is here and it is time to bloom."
by Jenni Sahagun
Bloom me Lord!


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Daffodil Software Limited hired IMSians!
IMS Ghaziabad feels delighted in sharing the success of its PGDM Students (2014-16).
Following Students of PGDM 2014-16 have been selected in Daffodil Software Limited.
1. Mr. Aditya Upadhyaya
2. Mr. M. Nithin
3. Mr. Nitish Bisht
4. Ms. Shriya Ranjan
5. Ms. Yashika Dua
Congratulations IMSians on successful placement !!!
You Make us PROUD !!
We would like to thank our prominent recruiters like Redington India, Axis Bank, Tata Tele Services, Pantaloons Retail - Aditya Birla Group, Hind Terminals, Ernst and Young, Deloitte, Nestle, American Express, S&P Capital IQ, Copal Amba, Grofers and many more for their support during campus recruitments.


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Cubus Velox game
Cubus Velox is a one-button, high-speed, extreme difficult platformer. Guide the Cubus through 30 levels filled with seemingly impassable passages and defy gravity while you're gliding through vast fields of spikes and traps. Featuring a retrotastic soundtrack by Eiswuxe! Also available on Android and iOS.
Cubus Velox, play free Mario games, related games and updates online. Added on
Author: knarrenheinz 23
Here are some more related games in Mario category. Also you can jump to other category using box above.
Related games
- Super Tiny Leap
- Flash Free Fall 1.5
- Neon Guy
- Lighting all the way
- Death Count
- RGB Shift
- Lusfod
- Splatz
- Clicktastic
- Casus
- Neon Force Runner
- Dungeon of Pain
- Spiked
- Linx Hard Levelset
- Hollow
- Orb Unlimited
- Scroll Lock
- Pixel Jump
- Stonelegs
- Pacmob
- Sleepwalker LD
- Loot Hero
- Bomb Blitz
- Impossible 2: Jungle Escape
- Michigan Hawk
- The Froginator
- Starship Operation Dark Matter
- Qobix


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Resources – Attention-Deficit Hyperactivity Disorder (ADHD)
Kaitlyn Atkinson Family Resource LibraryChildren's Hospital of Eastern Ontario Printer Friendly Version401 Smyth Rd., Ottawa, ON K1H email@example.com us @CHEOfrl General Information & Resources for ParentsResources for Kids & TeensResources for Learning, Schools & TeachersWebsitesLocal ContactsAppsGeneral Information & Resources for Parents
The ADD & ADHD answer book / Ashley, Susan -- Naperville, IL: Sourcebooks Inc., 2005.This is a reassuring, authoritative reference for you and your family, providing sound advice and immediate answers to your most pressing questions. The book also includes questionnaires and checklists to help you get the most out of your child's evaluation. FAM RJ 506 .H9 A327 2005 ADHD: a complete and authoritative guide -- [Elk Grove Village, IL] American Academy of Pediatrics, 2004.Based on the American Academy of Pediatrics' own clinical practice guidelines for ADHD and written in clear, accessible language, this book answers common questions, addresses behaviors associated with the teenage years and what schools can do to support children with the condition. ADHD management strategies that balance the roles of behavior therapy, medications, and parenting techniques are suggested. FAM RJ 506 .H9 A34 2004 ADHD: the facts -- Oxford: Oxford University Press, 2009. This book is intended to educate any interested party about ADHD. Both medical and non-medical persons will find it easy to understand. It is a very informative work written by a developmental pediatrician. FAM RJ 506 .H9 S448 2009 ADHD: what every parent needs to know -- Elk Grove, Ill.: American Academy of Pediatrics, 2011.This guide to attention deficit/hyperactivity disorder (ADHD) offers parents balanced, reassuring, and authoritative information to help them understand and manage this challenging and often misunderstood condition. It answers the common questions: How is ADHD diagnosed? What are today's best treatment options, and will my child outgrow ADHD?FAM RJ 506.H9 .A336 2011All about attention deficit disorder: symptoms, diagnosis and treatment: children and adults [DVD] / Phelan, Thomas W. -- Glen Ellyn, IL: Child Management Inc., 2005.This comprehensive resource give parents, teachers, pediatricians and mental health professionals the facts and resources they need to effectively deal with ADD. This program provides a thorough description of the symptoms, diagnosis and treatment of ADD FAM RJ 506 .H9 P44 2005 Apprivoiser l'hyperactivité et le déficit de l'attention / Sauve, Colette -- Montréal: Éditions de l'Hôpital Sainte-Justine, 2000.Comment gérer le comportement parfois étourdissant de votre enfant pour lequel un diagnostic d'hyperactivité ou de déficit de l'attention a été posé ? Voici proposée une gamme de moyens d'action dynamiques et diversifiés pour l'aider à s'épanouir dans sa famille, à l'école et dans son milieu de vie. L'auteur présente pour chaque groupe d'âge (3-5 ans, 6-12 ans, adolescence) trois parcours : 1) s'informer, comprendre, accepter ce désordre neurologique ; 2) prendre conscience de ses capacités d'éducateur ; 3) mettre en pratique de nouvelles stratégies. Pour multiplier les chances de réussite de votre enfant, ce guide vous invite enfin à croire en votre créativité et en votre intuition de parent. FAM RJ 506 .H9 S38 2000 Give your ADD teen a chance: a guide for parents of teenagers with attention deficit disorder / Weiss, Lynn -- Colorado Springs, CO: Pinon Press, 1996.This book examines the academic challenges ADD teens face, offering tips for success at school and guidelines for discipline, guidance, and responsibility. FAM RJ 506 .H9 W44 Helping hyperactive kids: a sensory integration approach: techniques and tips for parents and professionals / Horowitz, Lynn -- Alameda, CA: Hunter House Publishers, 2007. Sensory integration is a drug-free, child-friendly therapy for hyperactivity. This hands-on guide explains how it works and includes easy activities.FAM RJ 506 .H9 H6713 2007 Howard B. Wigglebottom blends in like chameleons: a fable about belonging / Binkow, Howard -- Thunderbolt Publishing, 2011. Howard helps his friend Joey understand that in order to overcome difficulties with attention, following instructions and finishing tasks, he needs to ask for professional help. Tips and lessons are included.FAM RJ 506 .H9 B56 2012Making the system work for your child with ADHD / Jensen, Peter S. -- New York: Guilford, 2004.In this straightforward, compassionate guide, Dr. Jensen pools his own experiences with those of over 80 other parents to help you troubleshoot the system without reinventing the wheel. From breaking through bureaucratic bottlenecks at school to advocating for your child's healthcare needs is just an example of topics discussed in this book. FAM RJ 506 .H9 J46 Medication for ADHD yes or no?: a practical guide [DVD] / Phelan, Thomas W. -- Glen Elly, IL: ParentMagic Inc., 2004.Discusses the pros and cons of medication for ADHD for both children and adults. FAM RJ 506 .H9 P442 2004 Only a mother could love him: my life with a triumph over ADD / Polis, Benjamin -- UK: Ballantine Books, 2004.This book will give you a look inside the mind of a person with ADD/ADHD. Ben describes what it's really like to feel those constant impulses, to get all that medication, to desperately want to be "normal." In addition, he offers lots of valuable advice to parents, including finding what forms of discipline will work and what will never work surviving the daily homework struggle medicating or seeking other methods of treatment teaching your ADD/ADHD child to read. FAM RJ 506 .H9 P64 2004Teenagers with ADD and ADHD: a guide for parents and professionals / Zeigler Dendy, Chris A. -- Bethesda, MD: Woodbine House, 2006.Provides insight, advice, and guidance to help parents effectively raise AD/HD teenagers.FAM RJ 506 .H9 Z45 2006 Le trouble de déficit de l'attention avec ou sans hyperactivité / Bélanger, Stacey [et al.] -- Montréal: Éditions du CHU Sainte-Justine, 2008. Ce livre est un ouvrage multidisciplinaire qui décrit l'ensemble des symptômes de TDAH et leur impact sur l'apprentissage de même que sur les relations familiales et sociales. Est également décrit le processus d'évaluation ainsi qu'une mise au point sur le traitement, tant sur le plan médicale qu'en ce qui concerne les interventions scolaires, comportementales et sociales. FAM RJ 506 .H9 T76 2008 Unlocking the potential of patients with ADHD: a model for clinical practice / Monastra, Vincent, J. -- Washington, DC: American Psychological Association, 2008. Written by a clinical researcher who has evaluated and treated over 10,000 patients with ADHD, this volume provides a foundation for translating the complex scientific literature on ADHD into a model for community-based care. FAM RJ 506 .H9 M654 2008 Visual perception problems in children with ADHD, autism and other learning disabilities: a guide for parents and professionals / Kurtz, Lisa A. -- London; Philadelphia: Jessica Kingsley Publishers, 2006. This book provides a comprehensive overview of vision problems in children with autism spectrum disorders and specific learning disabilities and outlines a range of activities for strengthening children's functional vision and perceptual skills using simple, homemade materials that are readably available in the home or classroom. This is an excellent practical companion for parents of children with visual perception problems and the professionals who work with them. FAM RJ 506 .H9 K87 2006 Back to TopResources for Kids & Teens
ADD and the college student: a guide for high school and college students with attention deficit disorder -- Washington, DC: Magination Press, 2001.Armed with the wisdom of specialists and those who have "been there" themselves, readers with ADD can approach the prospect of college with confidence. This concise handbook is packed with practical information and advice to help students navigate the smoothest possible transition to college life. FAM RJ 506 .H9 A33 2001 Adolescents and ADD: gaining the advantage / Quinn, Patricia O. -- New York: Magination Press, 1995. Addresses the particular concerns of adolescents diagnosed with attention deficit disorder and offers coping strategies as well as personal stories from teens with the disorder. FAM RJ 506 .H9 Q55 1995 All dogs have ADHD / Hoopman, Kathy -- London; Philadelphia: Jessica Kingsley Publishers, 2009. Contains pictures of dogs in usual and unusual positions help illustrate how the behaviours of people with attention-deficit hyperactivity disorder are similar to those of dogs. FAM RJ 506 .H9 H65 2009 Annie's plan: taking charge of schoolwork and homework / Kraus, Jeanne -- Washington, DC: Magination Press, 2007. With the help of her teacher and parents, Annie learns how to organize her desk, pay attention in class, and prepare her homework assignments. FAM RJ 506 .H9 K72 2007 Cory stories: a kid's book about living with ADHD / Kraus, Jeanne -- Washington, DC: Magination Press, 2005. A story about Cory, a young boy who has ADHD, who explains some of the difficulties he has at home and at school. Solutions are presented to make life easier. Appropriate for ages: 6-11 years.FAM RJ 506 .H9 K73 2005 Daydreaming Dakota: the world's greatest daydreamer / Bobula, Jill -- Nepean, ON: Wildberry Productions, 2008. This book is the story of a young girl who is affected by attention deficit disorder who describes her passion for people and countries around the world and the challenges she faces. FAM RJ 506 .H9 B64 2008 Eager Eddy: the world's most active dude: attention deficit hyperactivity disorder (ADHD) / Bobula, Jill -- Nepean, ON: Wildberry Productions, 2007.Eddy is portrayed as a happy-go-lucky kid who doesn't see his affliction with ADHD as a problem, but rather as something that makes him special. This book was written to help educate young people, teachers and even parents, that the diagnosis of a mental (or neurological) health disorder is not necessarily the end of the world, and with some help and understanding, they can work through it. FAM RJ 506 .H9 B66 2007 Lucky horseshoes: a tale from the Iris the dragon series: a children's book dealing with ADHD / Grass, Gayle -- Perth, ON: Iris the dragon, 2006. Skippy, a youngster with ADHD, overcomes her frustration with her difficulties at school and at home after meeting Iris, the Green Dragon. FAM RJ 506 .H9 G77 2006 Mom, why can't I catch up? / Simeton, Julia -- Bloomington, Indiana: AuthorHouse, 2009. The story about Joey J.J. Jones who doesn't fit in with other students because he has Attention Deficit Hyperactivity Disorder (ADHD). FAM RJ 506 .H9 S57 2009 Mon cerveau a besoin de lunettes: vivre avec l'hyperactivité / Vincent, Annick -- Le Gardeur, QC: Association Québécoise des Trouble D'Apprentissage, 2006.Ponctué de données instructives et de trucs efficaces, le journal imaginaire de Tom permet aux jeunes, aux parents et aux intervenants d'apprivoiser le trouble déficitaire de l'attention avec ou sans hyperactivité. Cette livre est indispensable pour mieux comprendre cette réalité. FAM RJ 506 .H9 V55 2006 My brain needs glasses: living with hyperactivity / Vincent, Annick -- Lac-Beauport, QC: Impact Academy Publishing, 2004.Filled with instructive information and effective hints, the imaginary journal of Tom helps children, parents, and education and psychology professionals overcome attention- deficit/hyperactivity disorder. Funny and imaginative, this book will help you understand the reality of kids living with AD/HD. FAM RJ 506 .H9 V5513 2004 Back to TopResources for Leaning, Schools & TeachersADHD in the schools: assessment and intervention strategies / Dupaul, George -- New York: Guilford Press, 2003.This book provides essential guidance for school-based professionals meeting the challenges of ADHD at any grade level. Comprehensive and practical, the book includes several reproducible assessment tools and handouts. FAM RJ 506 .H9 D87 2003 All about ADHD: the complete practical guide for classroom teachers / Pfiffner, Linda J. -- New York: Scholastic Professional Books, 1996.Provides teachers with knowledge about ADHD, and arms them with a variety of strategies for dealing with ADHD students. FAM RJ 506 .H9 P54 Back to TopWebsites
Helping Children and Youth with Attention Deficit and Hyperactivity Disorders – CHEO / Trouble d'hyperactivité avec deficit de l'attention – CHEOAttention Deficit Disorders – From the Canadian Mental Health Association / Les troubles déficitaires de l'attention – Association canadienne pour la santé mentaleADHD - MedlinePlus from the U.S. National Library of MedicineADHD - American Academy of Child & Adolescent PsychiatryChildren and Adults with Attention Deficit / Hyperactivity DisorderKids Health – Information for Kids, Teens and Parents from the Nemours FoundationCentre for ADD/ADHD Awareness Canada Les troubles d'hyperactivité avec déficit d'attention – Naître et Grandir Trouble déficitaire de l'attention avec ou sans hyperactivité – Site personnel D'Annick Vincent / Attention Deficit Disorder With or Without Hyperactivity (ADHD) – Personal Site of Annick VincentBack to TopLocal Contacts
Idao-c - Learning Disabilities Association of Ottawa/Carleton160 Percy StreetOttawa, ON K1R 6E5 email@example.com Canadian Mental Health Association – Ottawa Branch / Association canadienne pour la santé mentale – section Ottawa 301 - 1355 Bank Street Ottawa, ON K1H 8K7 Phone: 613-737-7791 Fax: 613-737-7644 email@example.com Association québécoise pour les troubles d'apprentissage (AQETA)109, rue Wright, bureau 203 Gatineau, Québec J8X 2G7Téléphone email@example.com
ADHD Tracker 1.0 (for Apple products) · American· From the American Academy of Pediatrics· English· Aimed at parents and teachers of children ages 4 through 18 who have already been diagnosed and treated for ADHD · This app is not a diagnostic tool. It can assist a healthcare team to monitor achild's progress with ADHD treatment in school and at home, helping them gain a better understanding of how ADHD is impacting the child's mental and physical health. This guide provides medical material for information purposes only and is not intended to replace the advice of your physician. The information may not always apply to your individual situation. The guide represents material available in the Family Resource Library collection at CHEO. It is not intended to be an exhaustive list. Your local public library may also be able to provide valuable information. If you require materials in alternative formats, please contact the library at 613-737-7600 ext. 2157 or at email@example.com. Last Updated: November 2015Links Tested: November 2015
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