No abstract

Molecular mechanisms underlying bladder dysfunction in ischemia, particularly at the protein and protein modification levels and downstream pathways, remain largely unknown. Here we describe a comparison of protein sequence variations in the ischemic and normal bladder tissues by measuring the mass differences of the coding amino acids and actual residues crossing the proteome. A large number of nonzero delta masses (11,056) were detected, spanning over 1295 protein residues. Clustering analysis identified 12 delta mass clusters that were significantly dysregulated, involving 30 upregulated (R2 > 0.5, ratio > 2, p < 0.05) and 33 downregulated (R2 > 0.5, ratio < −2, p < 0.05) proteins in bladder ischemia. These protein residues had different mass weights from those of the standard coding amino acids, suggesting the formation of non-coded amino acid (ncAA) residues in bladder ischemia. Pathway, gene ontology, and protein–protein interaction network analyses of these ischemia-associated delta-mass containing proteins indicated that ischemia provoked several amino acid variations, potentially post-translational modifications, in the contractile proteins and stress response molecules in the bladder. Accumulation of ncAAs may be a novel biomarker of smooth muscle dysfunction, with diagnostic potential for bladder dysfunction. Our data suggest that systematic assessment of global protein modifications may be crucial to the characterization of ischemic conditions in general and the pathomechanism of bladder dysfunction in ischemia.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate, which is the first committed step in the pathway for isoprenoid biosynthesis in plants. A full-length cDNA encoding HMGR (designated as EuHMGR, GenBank Accession No. AY796343) was isolated from Eucommia ulmoides by rapid amplification of cDNA ends (RACE). The full-length cDNA of EuHMGR comprises 2281 bp with a 1770-bp open reading frame (ORF) encoding a 590-amino-acid polypeptide with two trans-membrane domains revealed by bioinformatic analysis. Molecular modeling showed that EuHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. The deduced protein has an isoelectric point (pI) of 6.89 and a calculated molecular weight of about 63 kDa. Sequence comparison analysis showed that EuHMGR had highest homology to HMGR from Hevea brasiliensis. As expected, phylogenetic tree analysis indicated that EuHMGR belongs to plant HMGR group. Tissue expression pattern analysis showed that EuHMGR is strongly expressed in the leaves and stems whereas it is only poorly expressed in the roots, which implies that EuHMGR may be a constitutively expressing gene. Functional complementation of EuHMGR in HMGR-deficient mutant yeast JRY2394 demonstrated that EuHMGR mediates the mevalonate biosynthesis in yeast.

Background/Aims: This study investigated the effect of mechanical stress on tendon-bone healing in a rabbit anterior cruciate ligament (ACL) reconstruction model as well as cell proliferation and matrix formation in co-culture of bone-marrow mesenchymal stem cells (BMSCs) and tendon cells (TCs). Methods: The effect of continuous passive motion (CPM) therapy on tendon-bone healing in a rabbit ACL reconstruction model was evaluated by histological analysis, biomechanical testing and gene expressions at the tendon-bone interface. Furthermore, the effect of mechanical stretch on cell proliferation and matrix synthesis in BMSC/TC co-culture was also examined. Results: Postoperative CPM therapy significantly enhanced tendon-bone healing, as evidenced by increased amount of fibrocartilage, elevated ultimate load to failure levels, and up-regulated gene expressions of Collagen I, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin at the tendon-bone junction. In addition, BMSC/TC co-culture treated with mechanical stretch showed a higher rate of cell proliferation and enhanced expressions of Collagen I, Collagen III, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin than that of controls. Conclusion: These results demonstrated that proliferation and differentiation of local precursor cells could be enhanced by mechanical stimulation, which results in enhanced regenerative potential of BMSCs and TCs in tendon-bone healing.