Histone post-translational modifications (PTMs) are involved in diverse biological processes and methylation was regarded as a long-term epigenetic mark. Though aging represented one of the major risk factors for neurodegenerative diseases, no systematic investigations had correlated the patterns of histone PTMs in the brain with aging and the roles of such concerted histone PTMs in brain aging are still unknown. In this study, enzyme digestion, nano-LC, MALDI-TOF/TOF MS analysis and Western blotting were combined to investigate the defined methylation of core histones (H2A, H2B, H3 and H4) in the brain of 12-month-old senescence accelerated mouse prone 8 (SAMP8). The expression of several modified histones in the brain of 3-, and 12-month-old SAMP8 mice as well as that of the age-matched control senescence accelerated-resistant mouse (SAMR1) was compared. In the brain of 12-month-old SAMP8 mice, seven methylation sites (H3K24, H3K27, H3K36, H3K79, H3R128, H4K20 and H2A R89) were detected and most PTMs sites were located on histone H3. Mono-methylated H4K20 decreased significantly in the brain of 12-month-old SAMP8 mice. Methylated H3K27 and H3K36 coexisted in the aged brain with different methylation multiplicities. Di-methylated H3K79 expressed in the neurons of cerebral cortex and hippocampus. This study showed histone methylation patterns in the aged SAMP8 mice brain and provided the experimental evidences for further research on histone PTMs in the aged brain. We hope these results could initiate a platform for the exchange of comprehensive information concerning aging or neurodegenerative disease and help us interpret the change of gene expression and DNA repair ability at epigenetic level.

Phragmites communis Trin. (common reed) is a recognized model plant for studying its adaptation to contrasting and harsh environments. To understand the inherent molecular basis for its remarkable resistance to combined stresses, we performed a comprehensive proteomic analysis of the leaf proteins from two ecotypes, i.e. swamp and desert dune, naturally growing in the desert region of northwestern China. First, a proteome reference map of Phragmites was established based on the swamp ecotype. Proteins were resolved by 2-D/SDS-PAGE and identified by MALDI-TOF/TOF MS. In total, 177 spots were identified corresponding to 51 proteins. The major proteins identified are proteins involved in photosynthesis, glutathione and ascorbic acid metabolism as well as protein synthesis and quality control. Second, the 2-DE profiles of the two ecotypes were compared quantitatively via DIGE analysis. Compared with swamp ecotype, 51 proteins spots are higher-expressed and 58 protein spots are lower-expressed by twofold or more in desert dune ecotype. Major differences were found for the proteins involved in light reaction of photosynthesis, protein biosynthesis and quality control and antioxidative reactions. The physiological significance of such differences is discussed in the context of a flow of complex events in relation to plant adaptation to combined environmental stresses.

Methylation of lysine 20 in histone H4 has been proven to play important roles in chromatin structure and gene regulation. SET8 is one of the methyltransferases identified to be specific for this modification. In this study, the minimal active SET domain of SET8 has been mapped to the region of amino acids 195-352. This region completely retains the same methylation activity and substrate specificity as the full-length SET8. The SET domain recognizes a stretch of specific amino acid sequence around lysine 20 of H4 for its methylation activity. Methylation assays with N terminus mutants of H4 that contain deletions and single alanine or glutamine substitutions of charged residues revealed that SET8 requires the sequence RHRK 20 VLRDN for methylation at lysine 20. The individual mutation of any charged residue in this sequence to alanine or glutamine abolished or greatly decreased levels of methylation of lysine 20 of H4 by SET8. Interestingly, mutation of lysine 16 to alanine, arginine, glutamine, or methionine did not affect methylation of lysine 20 by the SET domain. Mass spectrometric analysis of synthesized H4 N-terminal peptides modified by SET8 showed that SET8 selectively mono-methylates lysine 20 of H4. Taken together, our results suggested that the coordination between the amino acid sequence RHRK 20 VLRDN and the SET domain of SET8 determines the substrate specificity and multiplicity of methylation of lysine 20 of H4.Chromosomal nucleosomes in eukaryotic cells consist of an octamer core of histones H2A, H2B, H3, and H4, as well as 146 bp of DNA, which wraps around the histone core. The majority of the histone protein residues fold inside the histone core, whereas their unstructured N-terminal tails stretch outside the nucleosome (1). Covalent modifications of histones, such as acetylation, methylation, and phosphorylation, have been demonstrated to play important roles in the regulation of chromatin structure and gene activity (2-8). Acetylation of histone lysines has been proven to be associated with euchromatin formation and maintenance, as well as gene activation (9, 10). Five lysine residues in the N termini of histones H3 and H4 have been identified to be the main target sites of methylation: lysines 4, 9, 27, and 36 of histone H3 and lysine 20 of histone H4. To date, methylation of H3 lysines has been extensively studied, and the role of tri-methylation of lysine 9 in heterochromatin formation and gene repression has been well documented (4, 6, 11-13). Structures of SET domains of several histone H3 methyltransferases have been elucidated (14 -21).Lysine 20 of H4 has been shown to be methylated to the mono-, di-, or tri-methylation forms in vivo (22-25). Human PR-Set7 and SET8 were first reported to be two methyltransferases responsible for methylation of lysine 20 of H4 (26 -28). Suv4-20h1 and Suv-20h2 were then identified to be specific for tri-methylation of lysine 20 of H4 (25). In yeast, Set9 was reported to be able to mono-, di-, and tri-methylate lysine 20 of H4 (29). Most methyltransferas...

Background: The S (spike) protein of the etiologic coronavirus (CoV) agent of severe acute respiratory syndrome (SARS) plays a central role in mediating viral infection via receptor binding and membrane fusion between the virion and the host cell. We focused on using synthetic peptides for developing antibodies against SARS-CoV, which aimed to block viral invasion by eliciting an immune response specific to the native SARS-CoV S protein.Methods: Six peptide sequences corresponding to the surface regions of SARS-CoV S protein were designed and investigated by use of combined bioinformatics and structural analysis. These synthetic peptides were used to immunize both rabbits and monkeys. Antisera collected 1 week after the second immunization were analyzed by ELISA and tested for antibody specificity against SARS-CoV by immunofluorescent confocal microscopy. Results: Four of our six synthetic peptides (S2, S3, S5, and S6) elicited SARS-CoV-specific antibodies, of which S5 (residues 788 -820) and S6 (residues 1002-1030) exhibited immunogenic responses similar to those found in a parallel investigation using truncated recombinant protein analogs of the SARS-CoV S protein. This

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