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Figure 4

-Deglycosylation of Epo-Epo fusion protein. Epo-Epo was treated with -glycanase, followed by SDS-PAGE and Western blotting (see “Experimental Procedures”). Note the narrow band of ∼42 kDa, consistent with full-deglycosylation. A minor band of ∼38–39 kDa is also visible and probably represents Epo-Epo lacking -linked sugars.

Epo-Epo is highly biologically active in vitro . We transfected COS1 cells with either conventional Epo wt cDNA or Epo-Epo (Epo wt /Epo wt ) cDNA. After 3 days, the supernatants were harvested and subjected to an in vitro bioassay. Protein was measured by ELISA calibrated against recombinant human Epo (Table bar strap sandals Grey Giuseppe Zanotti Cheapest 5V4PFKN8gM
). Epo wt was expressed at modest levels (3.5–9.2 IU/ml; 0.010–0.026 μg/ml) and exhibited a mean specific activity of 352 IU/μg (13 IU/pmol). This specific activity is slightly higher than that obtained by us previously for wild-type Epo expressed in COS1 cells (201–284 IU/μg) ( 10 ). In marked contrast, we found a much higher level of expression of Epo wt /Epo wt (110–195 IU/ml; 0.109–0.194 μg/ml), which exhibited a substantially higher mean specific activity (1007 IU/μg; 76 IU/pmol).

View this table:
Table I

Biological activities and protein concentrations of Epo and Epo-Epo fusion protein in transfected COS1 cell medium

Both Epo domains of Epo wt /Epo wt are biologically active. We showed this by mutation of arginine 103 (or the arginine 103 equivalent in Domain B, which is arginine 223 of the fusion protein) to alanine (R103A). This mutation results in complete inactivation of the Epo wt protein ( Outlet Find Great Black OverTheKnee Sock Boots Miu Miu New Fashion Style Of Sale 100% Guaranteed 2KAt5asH
, 20 ). We prepared three different constructs that incorporated this R103A mutation: ( a ) Epo R103A /Epo wt , ( b ) Epo wt /Epo R103A , and ( c ) Epo R103A /Epo R103A . We reasoned that if each of the two domains were active, then mutating only one domain would result in a protein that still retained biological activity, but with some decrease in specific activity. Only the double mutant Epo R103A /Epo R103A should be inactive. As seen in Table I , both Epo R103A /Epo wt and Epo wt /Epo R103A were expressed efficiently, and both exhibited biological activity. Interestingly, the mean specific activities of the two mutants were 480 (36 IU/pmol) and 516 IU/μg (39 IU/pmol), respectively, essentially one-half that of the non-mutated Epo wt /Epo wt (1007 IU/μg; 76 IU/pmol). This result strongly suggests that introduction of the R103A mutation into each Epo domain resulted in inactivation of that domain, allowing the other, wild-type domain to activate the receptor. This indicates that both Epo wt domains of Epo wt /Epo wt are biologically active, i.e. independently capable of receptor activation, and nearly equally so. The double mutant Epo R103A /Epo R103A was expressed at the mRNA level (data not shown). However, no activity or protein was detected in the COS1 supernatant.

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Notice of Meeting

Association of Environmental Engineering Geologists Date: Wednesday 11/16/2016 http://www.aegweb.org SECTION MEETING

Baltimore MD – Washington DC – Harrisburg PA

TOPIC: Geopier Rammed Aggregate Pier™ Applications Regional Case Studies

TOPIC: Geopier Rammed Aggregate Pier™ Applications Regional Case Studies

PRESENTER: Ms. Shana Carroll, P.E., Northeast Preconstruction Manager; GeoStructures, Inc.

PRESENTER: Shana Carroll, P.E., Northeast Preconstruction Manager; GeoStructures, Inc.

Rammed aggregate pier systems are a ground improvement technology that can be used to increase bearing capacity and control settlement of buildings, tanks, Retaining walls and embankments to close tolerances. Depending on site requirements, RAP systems can be installed using replacement (Geopier GP3®) or displacement (Geopier Impact®) methods. RAP systems are used to reinforce good to poor soils, including soft to stiff clay and silt; loose to dense sand; organic silt and peat; variable, uncontrolled fill; and soils below the ground water table. They can be used as an alternative to deep foundations or over-excavation, and provide cost and time savings on sites where poor soils and/or existing undocumented fill are a concern in terms of bearing capacity and settlement. This presentation reviews fundamental Geopier design and construction techniques and includes regional case studies such. Projects discussed include the National Cancer Institute’s Advanced Technology Research Facility located in Frederick Maryland and underlain by karst geology in the Piedmont Province, the Gaylord National River Ballroom located in National Harbor, MD in the Coastal Plain Province, and the George Mason University Fenwick Library in the Piedmont Province.

ABOUT THE PRESENTER:

Shana Carroll currently serves as Northeast Preconstruction Manager for GeoStructures, Inc. and was formerly an Engineer with GeoSyntec, Inc. Shana has been involved in the design, estimating, and pre-construction management of specialty geotechnical systems throughout the East Coast since 2001. Shana holds a B.S. M.S. degree in Civil and Environmental Engineering from Bucknell University and is a registered Professional Engineer in 5 states. She is the immediate past president of the Baltimore Chapter of American Society of Professional Estimators (ASPE) and on the Program Committee for the National Capital Section Geotechnical Executive Committee of the American Society of Civil Engineers (ASCE). In her spare time, Shana enjoys writing about topics related to young professionals on her blog ( www.outofthecube.net ) and traveling.

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Can judges and court staff assist with my case?

Court staff may explain basic procedures and answer questions about deadlines and fees. However, Iowa law and ethical rules prohibit judges and court staff from giving legal advice. This means that judges and staff cannot advise people on whether to bring claims to the court, what remedies they should seek, and the proper course of action.

Can I talk to the judge about my case without the other parties present?

No. Judicial ethics strictly prohibit judges from "ex parte" communication—which means talking with oneattorney or party without the otherattorney or party present.

If you want to give the judge informationabout yourcase or you want the judge to take some actionon yourcase, you must file your request with the court.

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Ask your friends, family, and colleagues for a referral or use the Iowa State Bar Association's Womens 26372 Trainers Marip Factory Price Outlet Store Locations Discount Recommend Outlet 2018 New Wholesale Price Cheap Price hOybeP1v
onlineor toll free: 1-800/532-1108.Court personnel are not permitted to make referrals to specific attorneys or law firms.

How do I request an ADA accommodation?

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Information on this web page is general in nature. Descriptions of laws and court procedures are abbreviated. This information is not intended as legal advice.If you do not understand this information or if you need legal advice you should seean attorney.

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