Influence of substitution pattern and cation binding on conformation and activity in heparin derivatives

As model compounds for the biologically important heparan sulfate, eight systematically modified heparin derivatives were studied by synchrotron radiation circular dichroism (SRCD), which is sensitive to uronic acid conformation. Substitution pattern altered uronic acid conformation, even when structural changes were made in adjacent glucosamine residues (e.g. 6-O-desulfation) and did not involve a chromophore. SRCD spectra of these derivatives following conversion to the Na+, K+, Mg2+, Ca2+, Mn2+, Cu2+ and Fe3+ cation forms revealed that almost all substitution/cation combinations resulted in unique spectra, showing that each was structurally distinct. The detailed effects that binding Na+, K+, Mg2+ and Ca2+ ions had on a 2-de-O-sulfated derivative was also studied by NMR, revealing that subtle changes in conformation (by NOE) and flexibility (by T2 measurements) resulted. Conversion to the K+ and Cu2+ ion forms also drastically modified biological activity, from inactive to active, in a cell-based assay of fibroblast growth factor-receptor (FGF2/FGFR1c) signalling and this effect was not reproduced by free cations. These observations could explain the often-contradictory data concerning structure-activity relationships for these derivatives in the literature and, furthermore, argue strongly against the established trend of considering sequence as a complete structural definition. It also provides additional means of modifying the activity of these polysaccharides and suggests a possible additional level of control in biological systems. There are also obvious potential applications for these findings in the biotechnology sphere.     

A systems biology approach for the investigation of the heparin/heparan sulfate interactome

A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.     

SSWAP: A Simple Semantic Web Architecture and Protocol for semantic web services

Background  

SSWAP (Simple Semantic Web Architecture and Protocol; pronounced "swap") is an architecture, protocol, and platform for using reasoning to semantically integrate heterogeneous disparate data and services on the web. SSWAP was developed as a hybrid semantic web services technology to overcome limitations found in both pure web service technologies and pure semantic web technologies.     

Pathologic diagnosis of breast cancer patients: evolution of the traditional clinical-pathologic paradigm toward “precision” cancer therapy

We present an updated account of breast cancer treatment and of progress toward “precision” cancer therapy; we focus on new developments in diagnostic molecular pathology and breast cancer that have emerged during the past 2 years. Increasing awareness of new prognostic and predictive methodologies, and introduction of next generation sequencing has increased understanding of both tumor biology and clinical behavior, which offers the possibility of more appropriate therapeutic choices. It remains unclear which of these testing methodologies provides the most informative and cost-effective actionable results for predictive and prognostic pathology. It is likely, however, that an integrated “step-wise” approach that uses the traditional clinical-pathologic paradigms coordinated with molecular characterization of breast tumor tissue, will offer the most comprehensive and cost-effective options for individualized, “precision” therapy for patients with breast cancer.     

Large-Scale Expression,Purification of Bioactive Recombinant Human FGF6 in <Emphasis Type="Italic">E. coli</Emphasis> and the Mechanisms of Its Myocardial Protection

Fibroblast growth factor 6 (FGF6) known as hst-2 belonging to the FGF4 subfamily has extensive biological activities which include regulating cell proliferation, angiogenesis, as well as facilitating muscle regeneration. To further investigate the structural and biochemical effects of FGF6, we had firstly established a successful E. coli system for large-scale production of recombinant human FGF6 (rhFGF6) with remarkable biological activity. We performed renaturation of denatured rhFGF6 on a SP Sepharose column followed by washing with buffer containing 600 mM NaCl, and further purification on a Heparin Sepharose column. The yield of purified rhFGF6 was nearly 110 mg per liter of bacterial broth and the purity of rhFGF6 was up to 97% which was demonstrated by HPLC analysis. In vitro studies showed that the rhFGF6 protein could significantly stimulate the proliferation of NIH 3T3 cells and C2C12 myoblast cells, besides protecting H9c2 myocardial cells against H₂O₂ induced injury through MAPK-Caspase-3 dependent pathway. The approach described here could provide an efficient avenue to produce active rhFGF6.     

Heparan sulfate in lung morphogenesis: The elephant in the room

Heparan sulfate (HS) is a structurally complex polysaccharide located on the cell surface and in the extracellular matrix, where it participates in numerous biological processes through interactions with a vast number of regulatory proteins such as growth factors and morphogens. HS is crucial for lung development; disruption of HS synthesis in flies and mice results in a major aberration of airway branching, and in mice, it results in neonatal death as a consequence of malformed lungs and respiratory distress. Epithelial–mesenchymal interactions governing lung morphogenesis are directed by various diffusible proteins, many of which bind to, and are regulated by HS, including fibroblast growth factors, sonic hedgehog, and bone morphogenetic proteins. The majority of research into the molecular mechanisms underlying defective lung morphogenesis and pulmonary pathologies, such as bronchopulmonary dysplasia and pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH), has focused on abnormal protein expression. The potential contribution of HS to abnormalities of lung development has yet to be explored to any significant extent, which is somewhat surprising given the abnormal lung phenotype exhibited by mutant mice synthesizing abnormal HS. This review summarizes our current understanding of the role of HS and HS‐binding proteins in lung morphogenesis and will present in vitro and in vivo evidence for the fundamental importance of HS in airway development. Finally, we will discuss the future possibility of HS‐based therapeutics for ameliorating insufficient lung growth associated with lung diseases such as CDH. Birth Defects Research (Part C) 90:32–44, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis of basic fibroblast growth factor upon differentiation of rat mammary epithelial to myoepithelial-like cells in culture

Acidic fibroblast growth factor (aFGF) mRNA was detected in a rat mammary fibroblastic cell line, but not in rat mammary epithelial cell lines or myoepithelial-like cell lines. Basic FGF (bFGF) mRNA was detected in both the fibroblasts and the myoepithelial-like cells, but was absent from the epithelial cells. A series of cell lines representing stages in the differentiation pathway of epithelial cells to a myoepithelial-like morphology showed an increase in the amount of bFGF mRNA and activity present and the FGF from the myoepithelial-like rat mammary 29 cells was able to displace [125I]-bFGF specifically bound to rat mammary fibroblasts. FGF activity was also present in an extract of rat mammary gland. Analysis of cell extracts and conditioned medium indicated that FGF activity was cell-associated. The cell-associated bFGF was resistant to degradation by trypsin. Extraction of myoepithelial-like cells with Triton X-100 and 2 M NaCl showed that 50-65% of the cell-associated bFGF was in a detergent-resistant but 2 M NaCl-labile structure. Thus, the synthesis of bFGF is developmentally regulated in rat mammary cell lines, and at least 50% is present in the extracellular matrix.     

Functions of exogenous FGF signals in regulation of fibroblast to myofibroblast differentiation and extracellular matrix protein expression

Fibroblasts are widely distributed cells found in most tissues and upon tissue injury, they are able to differentiate into myofibroblasts, which express abundant extracellular matrix (ECM) proteins. Overexpression and unordered organization of ECM proteins cause tissue fibrosis in damaged tissue. Fibroblast growth factor (FGF) family proteins are well known to promote angiogenesis and tissue repair, but their activities in fibroblast differentiation and fibrosis have not been systematically reviewed. Here we summarize the effects of FGFs in fibroblast to myofibroblast differentiation and ECM protein expression and discuss the underlying potential regulatory mechanisms, to provide a basis for the clinical application of recombinant FGF protein drugs in treatment of tissue damage.     

The cooperation of FGF receptor and Klotho is involved in excretory canal development and regulation of metabolic homeostasis in Caenorhabditis elegans

FGFs have traditionally been associated with cell proliferation, morphogenesis, and development; yet, a subfamily of FGFs (FGF19, -21, and -23) functions as hormones to regulate glucose, lipid, phosphate, and vitamin D metabolism with impact on energy balance and aging. In mammals, Klotho and beta-Klotho are type 1 transmembrane proteins that function as obligatory co-factors for endocrine FGFs to bind to their cognate FGF receptors (FGFRs). Mutations in Klotho/beta-Klotho or fgf19, -21, or -23 are associated with a number of human diseases, including autosomal dominant hypophosphatemic rickets, premature aging disorders, and diabetes. The Caenorhabditis elegans genome contains two paralogues of Klotho/beta-Klotho, klo-1, and klo-2. klo-1 is expressed in the C. elegans excretory canal, which is structurally and functionally paralogous to the vertebrate kidney. KLO-1 associates with EGL-15/FGFR, suggesting a role for KLO-1 in the fluid homeostasis phenotype described previously for egl-15/fgfr mutants. Altered levels of EGL-15/FGFR signaling lead to defects in excretory canal development and function in C. elegans. These results suggest an evolutionarily conserved function for the FGFR-Klotho complex in the development of excretory organs such as the mammalian kidney and the worm excretory canal. These results also suggest an evolutionarily conserved function for the FGFR-Klotho axis in metabolic regulation.