Differential Proteome Analysis of Bone Marrow Mesenchymal Stem Cells from Adolescent Idiopathic Scoliosis Patients

Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine. The cause and pathogenesis of scoliosis and the accompanying generalized osteopenia remain unclear despite decades of extensive research. In this study, we utilized two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) to analyze the differential proteome of bone marrow mesenchymal stem cells (BM-MSCs) from AIS patients. In total, 41 significantly altered protein spots were detected, of which 34 spots were identified by MALDI-TOF/TOF analysis and found to represent 25 distinct gene products. Among these proteins, five related to bone growth and development, including pyruvate kinase M2, annexin A2, heat shock 27 kDa protein, γ-actin, and β-actin, were found to be dysregulated and therefore selected for further validation by Western blot analysis. At the protein level, our results supported the previous hypothesis that decreased osteogenic differentiation ability of MSCs is one of the mechanisms leading to osteopenia in AIS. In summary, we analyzed the differential BM-MSCs proteome of AIS patients for the first time, which may help to elucidate the underlying molecular mechanisms of bone loss in AIS and also increase understanding of the etiology and pathogenesis of AIS

Comparative proteomic analysis reveals differential expression of Hsp25 following the directed differentiation of mouse embryonic stem cells

AbstractMurine embryonic stem (ES) cells can be committed to neural differentiation with high efficiency in culture through the use of feeder- and serum-free media. This system is proving to be an excellent model to study processes involved in ES cell commitment to neural cell fate. We used this approach to generate neurogenic embryoid bodies (NEBs) in a serum-free culture system to perform proteomic analysis of soluble fractions and identify early changes in protein expression as ES cells differentiate. Ten candidate proteins were altered significantly in expression levels. One of the most significant alterations was for the small heat shock protein Hsp25. Three species of Hsp25 are detected in ES cells, and this expression pattern changes during the first 24 h of differentiation until expression is decreased to levels that are barely detectable at 4 days following differentiation. We used immunofluorescence studies to confirm that following ES cell differentiation, expression of Hsp25 becomes excluded from neural precursors as well as other differentiating cells, making it a potentially useful marker of early ES cell differentiation

Sequential and Selective Buchwald-Hartwig Amination Reactions for the Controlled Functionalization of 6-Bromo-2-chloroquinoline: Synthesis of Ligands for the Tec Src Homology 3 Domain

Src homology 3 (SH3) domains are highly conserved protein-protein interaction domains that mediate important biological processes and are considered valuable targets for the development of therapeutic agents. In this paper, we report the preparation of a range of new 6-heterocyclic substituted 2-aminoquinolines using Buchwald-Hartwig chemistry. 6-Heterocyclic substitution of the 2-aminoquinoline has provided ligands with increased binding affinity for the SH3 domain relative to the lead compound, 2-aminoquinoline, that are the highest affinity ligands prepared to date. The key step in the synthesis of these compounds required a selective Buchwald-Hartwig amination of an aryl bromide in the presence of an activated heteroaryl chloride. The optimization of reaction conditions to achieve the selective amination is discussed and has allowed for cross-coupling with a range of cyclic amines. Introduction of the amino functionality of the 6-heterocyclic 2-aminoquinolines involved additional Buchwald-Hartwig chemistry utilizing lithium bis(trimethylsilyl)amide as an ammonia equivalent.Jessica A. Smith, Rhiannon K. Jones, Grant W. Booker and Simon M. Pyk