Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

Characterizing the metabolic phenotype: a phenotype phase plane analysis.

Genome-scale metabolic maps can be reconstructed from annotated genome sequence data, biochemical literature, bioinformatic analysis, and strain-specific information. Flux-balance analysis has been useful for qualitative and quantitative analysis of metabolic reconstructions. In the past, FBA has typically been performed in one growth condition at a time, thus giving a limited view of the metabolic capabilities of a metabolic network. We have broadened the use of FBA to map the optimal metabolic flux distribution onto a single plane, which is defined by the availability of two key substrates. A finite number of qualitatively distinct patterns of metabolic pathway utilization were identified in this plane, dividing it into discrete phases. The characteristics of these distinct phases are interpreted using ratios of shadow prices in the form of isoclines. The isoclines can be used to classify the state of the metabolic network. This methodology gives rise to a "phase plane" analysis of the metabolic genotype-phenotype relation relevant for a range of growth conditions. Phenotype phase planes (PhPPs) were generated for Escherichia coli growth on two carbon sources (acetate and glucose) at all levels of oxygenation, and the resulting optimal metabolic phenotypes were studied. Supplementary information can be downloaded from our website (http://epicurus.che.udel.edu).     

Changes in cholesterol levels in the plasma membrane modulate cell signaling and regulate cell adhesion and migration on fibronectin

The number and distribution of lipid molecules, including cholesterol in particular, in the plasma membrane, may play a key role in regulating several physiological processes in cells. We investigated the role of membrane cholesterol in regulating cell shape, adhesion and motility. The acute depletion of cholesterol from the plasma membrane of cells that were well spread and motile on fibronectin caused the rounding of these cells and decreased their adhesion to and motility on fibronectin. These modifications were less pronounced in cells plated on laminin, vitronectin or plastic, indicating that cholesterol-mediated changes in adhesion and motility are more specific for adhesion mediated by fibronectin-specific integrins, such as alpha5beta1. These changes were accompanied by remodeling of the actin cytoskeleton, the spatial reorganization of paxillin in the membrane, and changes to the dynamics of alpha5 integrin and paxillin-rich focal adhesions. Levels of tyrosine phosphorylation at position 576/577 of FAK and Erk1/Erk2 MAP-kinase activity levels were both lower in cholesterol-depleted than in control cells. These levels normalized only on fibronectin when cholesterol was reincorporated into the cell membrane. Thus, membrane cholesterol content has a specific effect on certain signaling pathways specifically involved in regulating cell motility on fibronectin and organization of the actin cytoskeleton.     

Novel three missense mutations observed in Von Hippel-Lindau gene in a patient reported with renal cell carcinoma

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumors, especially cerebellar hemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). We have identified of VHL gene using immunohistochemistry in a patient who was diagnosed for RCC. In order to understand the involvement of mutation in the VHL gene exon 1 was amplified and sequenced (accession number: JX 401534). The sequence analysis revealed the presence of novel missense mutations c.194 C>T, c.239 G>A, c.278 G>A, c.319 C>G, c. 337 C > G leading to the following variations p.Ala 65 Val, p.Gly 80 Asp, p.Gly 93 Glu, p.Gln 107 Glu, p.Gln 113 Glu in the protein.     

Antiproliferative and Apoptotic Effects of pH-Responsive Veratric Acid-Loaded Polydopamine Nanoparticles in Human Triple Negative Breast Cancer Cells

Veratric acid (VA) is plant-derived phenolic acid known for its therapeutic potential, but its anticancer effect on highly invasive triple-negative breast cancer (TNBC) is yet to be evaluated. Polydopamine nanoparticles (nPDAs) were chosen as the drug carrier to overcome VA's hydrophobic nature and ensure a sustained release of VA. We prepared pH-sensitive nano-formulations of VA-loaded nPDAs and subjected them to physicochemical characterization and in vitro drug release studies, followed by cell viability and apoptotic assays on TNBC cells (MDA-MB-231 cells). The SEM and zeta analysis revealed spherical nPDAs were uniform size distribution and good colloidal stability. In vitro drug release from VA-nPDAs was sustained, prolonged and pH-sensitive, which could benefit tumor cell targeting. MTT and cell viability assays showed that VA-nPDAs (IC50=17.6 μM) are more antiproliferative towards MDA-MB-231 cells than free VA (IC50=437.89 μM). The induction of early and late apoptosis by VA-nPDAs in the cancer cells was identified using annexin V and dead cell assay. Thus, the pH response and sustained release of VA from nPDAs showed the potential to enter the cell, inhibit cell proliferation, and induce apoptosis in human breast cancer cells, indicating the anticancer potential of VA.     

A 32 kb critical region excluding Y402H in CFH mediates risk for age-related macular degeneration

Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10⁻¹⁰⁹); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10⁻⁹) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.     

Recessive Mutations in SLC38A8 Cause Foveal Hypoplasia and Optic Nerve Misrouting without Albinism

Foveal hypoplasia and optic nerve misrouting are developmental defects of the visual pathway and only co-occur in connection with albinism; to date, they have only been associated with defects in the melanin-biosynthesis pathway. Here, we report that these defects can occur independently of albinism in people with recessive mutations in the putative glutamine transporter gene SLC38A8. Nine different mutations were identified in seven Asian and European families. Using morpholino-mediated ablation of Slc38a8 in medaka fish, we confirmed that pigmentation is unaffected by loss of SLC38A8. Furthermore, by undertaking an association study with SNPs at the SLC38A8 locus, we showed that common variants within this gene modestly affect foveal thickness in the general population. This study reveals a melanin-independent component underpinning the development of the visual pathway that requires a functional role for SLC38A8.     

Nonproteolytic properties of murine alternatively spliced tissue factor: implications for integrin-mediated signaling in murine models

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.