ESTIMATING RELATEDNESS USING GENETIC MARKERS

A new method is described for estimating genetic relatedness from genetic markers such as protein polymorphisms. It is based on Grafen's (1985) relatedness coefficient and is most easily interpreted in terms of identity by descent rather than as a genetic regression. It has several advantages over methods currently in use: it eliminates a downward bias for small sample sizes; it improves estimation of relatedness for subsets of population samples; and it allows estimation of relatedness for a single group or for a single pair of individuals. Individual estimates of relatedness tend to be highly variable but, in aggregate, can still be very useful as data for nonparametric tests. Such tests allow testing for differences in relatedness between two samples or for correlating individual relatedness values with another variable.     

Hepatoma-Derived Growth Factor-Related Protein-3 Is a Novel Angiogenic Factor

Hepatoma-derived growth factor-related protein-3 (Hdgfrp3 or HRP-3) was recently reported as a neurotrophic factor and is upregulated in hepatocellular carcinoma to promote cancer cell survival. Here we identified HRP-3 as a new endothelial ligand and characterized its in vitro and in vivo functional roles and molecular signaling. We combined open reading frame phage display with multi-round in vivo binding selection to enrich retinal endothelial ligands, which were systematically identified by next generation DNA sequencing. One of the identified endothelial ligands was HRP-3. HRP-3 expression in the retina and brain was characterized by Western blot and immunohistochemistry. Cell proliferation assay showed that HRP-3 stimulated the growth of human umbilical vein endothelial cells (HUVECs). HRP-3 induced tube formation of HUVECs in culture. Wound healing assay indicated that HRP-3 promoted endothelial cell migration. HRP-3 was further confirmed for its in vitro angiogenic activity by spheroid sprouting assay. HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells. The angiogenic activity of HRP-3 was independently verified by mouse cornea pocket assay. Furthermore, in vivo Matrigel plug assay corroborated HRP-3 activity to promote new blood vessel formation. These results demonstrated that HRP-3 is a novel angiogenic factor.     

Effects of introducing cultured human chondrocytes into a human articular cartilage explant model

Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. We have investigated the ability of implanted chondrocytes to attach at the site of injury and to be incorporated into the decellularized host matrix adjacent to a defect in an in vitro human explant model. Human osteochondral dowels received a standardized injury, were seeded with passage 3 chondrocytes labelled with PKH 26 and compared with two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Seeded chondrocytes adhered to the standardized defect and areas of lamina splendens disruption but did not migrate into the adjacent acellular region. A difference was noted in viable-cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present. The results indicate that cultured human chondrocytes do indeed adhere to regions of AC matrix injury but do not migrate into the host tissue, despite the presence of viable cells. This human explant model is thus an effective tool for studying the interaction of implanted cells and host tissue.     

Novel pyrrolyl 2-aminopyridines as potent and selective human β-secretase (BACE1) inhibitors

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid in Alzheimer’s disease. Recently, we reported small molecule acylguanidines as potent BACE1 inhibitors. However, many of these acylguanidines have a high polar surface area (e.g. as measured by the topological polar surface area or TPSA), which is unfavorable for crossing the blood–brain barrier. Herein, we describe the identification of the 2-aminopyridine moiety as a bioisosteric replacement of the acylguanidine moiety, which resulted in inhibitors with lower TPSA values and superior brain penetration. X-ray crystallographic studies indicated that the 2-aminopyridine moiety interacts directly with the catalytic aspartic acids Asp32 and Asp228 via a hydrogen-bonding network.     

Purification and <Emphasis Type="Italic">N</Emphasis>-Glycosylation Analysis of Melanoma Antigen Dopachrome Tautomerase

Dopachrome tautomerase (DCT) plays a critical role in lowering the oxidative stress resulting from melanogenesis. Levels of DCT are elevated in melanoma cell lines that are especially resistant to chemotherapy and radiation. DCT is processed as a melanoma antigen and is a potential target for immunotherapy. In order to establish a more complete understanding of the role that DCT may play in the etiology and treatment of melanoma skin cancer, isolation of highly pure and properly processed protein is necessary. Purification of native DCT has been problematic due to a hydrophobic transmembrane anchor and interactions with melanin. In this study, DCT was expressed, without its carboxy-terminal transmembrane region using an Sf9 insect cell protein expression system and its recombinant protein was purified by various chromatographic techniques. Analysis of DCT tryptic peptides by MALDI-TOF/TOF determined N-glycosylation as a primary post-translational modification. Our success in the expression of soluble mammalian DCT and the characterization of N-glycosylation sites is a useful reference toward the comprehensive understanding of the structure/function relationship of mammalian DCT.