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Proteomic Retrieval from Nucleic Acid Depleted Space-Flown Human Cells
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Author and Affiliation:
Hammond, D. K.(Enterprise Advisory Services, Inc., Houston, TX, United States);
Elliott, T. F.(Wyle Life Sciences, Inc., Houston, TX, United States);
Holubec, K.(Wyle Life Sciences, Inc., Houston, TX, United States);
Baker, T. L.(Wyle Life Sciences, Inc., Houston, TX, United States);
Allen, P. L.(Tulane Univ. Health Sciences Center, V.A. Medical Center, New Orleans, LA, United States);
Hammond, T. G.(Tulane Univ. Health Sciences Center, V.A. Medical Center, New Orleans, LA, United States);
Love, J. E.(NASA Johnson Space Center, Houston, TX, United States)
Abstract: Compared to experiments utilizing humans in microgravity, cell-based approaches to questions about subsystems of the human system afford multiple advantages, such as crew safety and the ability to achieve statistical significance. To maximize the science return from flight samples, an optimized method was developed to recover protein from samples depleted of nucleic acid. This technique allows multiple analyses on a single cellular sample and when applied to future cellular investigations could accelerate solutions to significant biomedical barriers to human space exploration. Cell cultures grown in American Fluoroseal bags were treated with an RNA stabilizing agent (RNAlater - Ambion), which enabled both RNA and immunoreactive protein analyses. RNA was purified using an RNAqueous(registered TradeMark) kit (Ambion) and the remaining RNA free supernatant was precipitated with 5% trichloroacetic acid. The precipitate was dissolved in SDS running buffer and tested for protein content using a bicinchoninic acid assay (1) (Sigma). Equal loads of protein were placed on SDS-PAGE gels and either stained with CyproOrange (Amersham) or transferred using Western Blotting techniques (2,3,4). Protein recovered from RNAlater-treated cells and stained with protein stain, was measured using Imagequant volume measurements for rectangles of equal size. BSA treated in this way gave quantitative data over the protein range used (Fig 1). Human renal cortical epithelial (HRCE) cells (5,6,7) grown onboard the International Space Station (ISS) during Increment 3 and in ground control cultures exhibited similar immunoreactivity profiles for antibodies to the Vitamin D receptor (VDR) (Fig 2), the beta isoform of protein kinase C (PKC ) (Fig 3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig 4). Parallel immunohistochemical studies on formalin-fixed flight and ground control cultures also showed positive immunostaining for VDR and other biomarkers (Fig 5). These results are consistent with data from additional antigenic recovery experiments performed on human Mullerian tumor cells cultured in microgravity (8).
Publication Date: Jan 01, 2006
Document ID:
20080026350
(Acquired Jul 29, 2008)
Subject Category: AEROSPACE MEDICINE
Document Type: Journal Article
Contract/Grant/Task Num: NAS9-02078; NAG8-1362
Financial Sponsor: NASA Marshall Space Flight Center; Huntsville, AL, United States
NASA Johnson Space Center; Houston, TX, United States
Organization Source: NASA Johnson Space Center; Houston, TX, United States
Description: 2p; In English; Original contains color and black and white illustrations
Distribution Limits: Unclassified; Publicly available; Unlimited
Rights: Copyright; Distribution as joint owner in the copyright
NASA Terms: CELLS (BIOLOGY); SPACEBORNE EXPERIMENTS; PROTEOME; INTERNATIONAL SPACE STATION; MICROGRAVITY; NUCLEIC ACIDS; CULTURED CELLS; ANTIBODIES; SPACE EXPLORATION; STATISTICAL ANALYSIS; SAFETY FACTORS; PROTEINS; STABILIZATION
Miscellaneous Notes: To be published in the Gravitational and Space Biology Bulletin, June 2006
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