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Biochemical characterization of mouse brain necdin.

ABSTRACT: Necdin is a protein encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells (P19). Necdin of mouse brain was characterized by Western blotting and silver-staining analysis by using affinity purified antibodies to 17 synthetic peptides of deduced C-terminal amino acids. Necdin exhibits a molecular mass of 51 kDa on SDS/PAGE, and is localized in the S1 and S2 nucleosomal fractions. Sonicated necdin is found in all fractions of Sephacryl S-300 gel filtration chromatography, with a peak at 700 kDa. Necdin is released on microsomal nuclease digestion, which is essential for electrophoretic migration on acetic acid/urea/Triton gels, suggesting that it could be a DNA-binding protein. Nucleosomal necdin shows two peaks at approx. 10 S and approx. 20 S on sucrose gradient centrifugation in the presence of 0.6 M NaCl, and a single peak in the presence of 2.0 M NaCl. Necdin forms a huge complex through chemical cross-linking with glutaraldehyde or dimethyl sulphate. The silver-staining intensity of the 51 kDa band corresponds to the decrease in the immuno-staining in a reagent concentration-dependent manner. Necdin binds tightly to a double-stranded DNA affinity chromatography column, and can be eluted from it with 2.0 M NaCl after washing with 0.6 M NaCl (approx. 100 ng per ml of gel). This purified necdin exhibits of pI of 9.1 on isoelectric focusing. The nucleosomal necdin complex (>200 kDa) was adsorbed on an organomercurial agarose affinity chromatography column and was eluted with 10 mM DTT, revealing that necdin is possibly involved in the transactive nucleosomal complex. These data show that necdin is a nuclear basic DNA-binding protein that associates with other molecules to regulate transcriptionally active genes and nuclear function.

SUBMITTER: Maruyama E

PROVIDER: S-EPMC1217141 | biostudies-other | 1996 Mar

REPOSITORIES: biostudies-other

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Project description:Repetitive mild traumatic brain injury (r-mTBI) results in neuropathological and biochemical consequences in the human visual system. Using a recently developed mouse model of r-mTBI, with control mice receiving repetitive anesthesia alone (r-sham) we assessed the effects on the retina and optic nerve using histology, immunohistochemistry, proteomic and lipidomic analyses at 3 weeks post injury. Retina tissue was used to determine retinal ganglion cell (RGC) number, while optic nerve tissue was examined for cellularity, myelin content, protein and lipid changes. Increased cellularity and areas of demyelination were clearly detectable in optic nerves in r-mTBI, but not in r-sham. These changes were accompanied by a ~25% decrease in the total number of Brn3a-positive RGCs. Proteomic analysis of the optic nerves demonstrated various changes consistent with a negative effect of r-mTBI on major cellular processes like depolymerization of microtubules, disassembly of filaments and loss of neurons, manifested by decrease of several proteins, including neurofilaments (NEFH, NEFM, NEFL), tubulin (TUBB2A, TUBA4A), microtubule-associated proteins (MAP1A, MAP1B), collagen (COL6A1, COL6A3) and increased expression of other proteins, including heat shock proteins (HSP90B1, HSPB1), APOE and cathepsin D. Lipidomic analysis showed quantitative changes in a number of phospholipid species, including a significant increase in the total amount of lysophosphatidylcholine (LPC), including the molecular species 16:0, a known demyelinating agent. The overall amount of some ether phospholipids, like ether LPC, ether phosphatidylcholine and ether lysophosphatidylethanolamine were also increased, while the majority of individual molecular species of ester phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were decreased. Results from the biochemical analysis correlate well with changes detected by histological and immunohistochemical methods and indicate the involvement of several important molecular pathways. This will allow future identification of therapeutic targets for improving the visual consequences of r-mTBI.

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Biochemical characterization of mouse brain necdin.

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