Effect of Endothelin-1 on proliferation,migration and fibrogenic gene expression in human RPE cells

The pathology of the fibrotic proliferative vitreoretinopathy (PVR) membrane represents an excessive wound healing response characterised by cells’ proliferation, migration and secretion of extracellular matrix molecules (ECMs). Retinal pigment epithelial (RPE) cells are a major cellular component of the fibrotic membrane. Endothelin-1 (ET-1) has been reported to be involved in the development of PVR in vivo research. However, little is known about the role of ET-1 in RPE cells in vitro. In the present study, we investigated the role of ET-1 in the proliferation, migration and secretion of ECMs (such as type I collagen and fibronectin) in RPE cells in vitro. Our results illustrated that ET-1 promoted the proliferation, migration and secretion of ECMs through the protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) signaling pathways in RPE cells in vitro. These findings strongly suggested that ET-1 may play a vital role in the development of PVR.     

Fibronectin (FN) in hypertrophic scars and keloids

Summary Fibronectin (FN) distribution was compared among samples of normal human dermis, hypertrophic scar, keloid, and granulation tissues from deep injuries. Localization was established by use of fibronectin antibodies and the indirect immunofluorescence method. Fresh-frozen tissue was sectioned on a cryostat and examined by epifluorescence. Hypertrophic scar and keloid demonstrated heavy deposition of FN, which conformed to the nodular characteristics of the lesions. Intense localization occurred in granulation tissue over fibroblasts which were stellate and vesiculated, and over small blood vessels. FN-staining was weak in areas over fibroblasts which were more rounded and nonvesiculated. Staining for FN was also minimal over the collagen in normal dermis and the deeper, larger collagen fascicles in the lesions. Fibroblasts cultured from normal dermis, hypertrophic scar, and keloid for 5–6 weeks were intensely stained for FN. Extracellular matrix was heavily positive in cultures from the lesions compared with those from normal dermis.Supported in part by NIH Research Grant 1 R01GM 25159     

The Hidden Cell-to-Cell Trail of α-Synuclein Aggregates

The progressive accumulation of insoluble aggregates of the presynaptic protein alpha-synuclein (α-Syn) is a hallmark of neurodegenerative disorders including Parkinson's disease (PD), Multiple System Atrophy, and Dementia with Lewy Bodies, commonly referred to as synucleinopathies. Despite considerable progress on the structural biology of these aggregates, the molecular mechanisms mediating their cell-to-cell transmission, propagation, and neurotoxicity remain only partially understood. Numerous studies have highlighted the stereotypical spatiotemporal spreading of pathological α-Syn aggregates across different tissues and anatomically connected brain regions over time. Experimental evidence from various cellular and animal models indicate that α-Syn transfer occurs in two defined steps: the release of pathogenic α-Syn species from infected cells, and their uptake via passive or active endocytic pathways. Once α-Syn aggregates have been internalized, little is known about what drives their toxicity or how they interact with the endogenous protein to promote its misfolding and subsequent aggregation. Similarly, unknown genetic factors modulate different cellular responses to the aggregation and accumulation of pathogenic α-Syn species. Here we discuss the current understanding of the molecular phenomena associated with the intercellular spreading of pathogenic α-Syn seeds and summarize the evidence supporting the transmission hypothesis. Understanding the molecular mechanisms involved in α-Syn aggregates transmission is essential to develop novel targeted therapeutics against PD and related synucleinopathies.     

Potential of mean force and molecular dynamics study on the transient interactions between α and β synuclein that drive inhibition of α-synuclein aggregation

Self-association of α-synuclein (αS) into pathogenic oligomeric species and subsequent formation of highly ordered amyloid fibrils is linked to the Parkinson’s disease. So most of the recent studies are now focused on the development of potential therapeutic strategies against this debilitating disease. β-synuclein (βS), a presynaptic protein that co-localizes with αS has been recently reported to act as an inhibitor of αS self-assembly. But the specificity of molecular interaction, nature and location between αS/βS is not known despite the potential importance of βS as an inhibitor of αS. We used molecular dynamics and potential of mean force (PMF) to study association of αS/βS and αS/αS. The calculated PMF indicates that contact wells are significantly deeper and presence of a minimum at αS/βS separation of 13.5 Å with a free energy barrier of 40 kcal/mol. We observed the dissociation energy barrier to be two times higher for the hetero-dimer (αS/βS) than the homo-dimer (αS/αS). We also carried out umbrella samplings involving two degrees of freedom (one being the distance between the monomeric units and the other angle between the long axes of the two monomeric chains) and observed similar PMF profile. We noticed relatively stronger range of transient interactions between the monomeric units in hetero-dimer (αS/βS) than homo-dimer (αS/αS). So our findings suggest that αS readily combines with βS to form hetero-dimer than combining with itself in forming homo-dimer. Hence we see predominant transient interactions between αS and βS can be used to drive inhibition of αS aggregation.     

Amphibian Pleurodeles waltl Fibronectin: cDNA Cloning and Developmental Expression of Spliced Variants

Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3′ region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.     

Bionanotechnology application of polypeptides in a hair color product: Self-assembly enables expression,processing, and functionality

Bionanotechnology aims to impart new properties to materials from unique functionalities present in biomolecules. However, the promise of bionanotechnology has not materialized beyond the biomedical field due in large part to issues of scalability, purity, and cost of manufacturing. In this work we demonstrate an approach to co-engineer production and system functionality into a single polypeptide. We designed a system to anchor particles onto hair via a multifunctional polypeptide composed of two domains, one with affinity to hair and the other capable of strong interactions with the particle surface. These strong interactions, exemplified by resistance to anionic surfactants, stem from the ability to self-assemble into higher order structures, which were observed by atomic force microscopy. At the same time, the controlled solubility properties of the particle binding domain permit the scalable production in Escherichia coli via inclusion bodies and cost effective purification. We believe this is a significant advance toward the development of bionanotechnology for industrial applications.     

Phosphorylation of fibronectin in quiescent and growing cell cultures

Phosphorylation of fibronectin was studied in quiescent vs growing cells from several species. Fibronectin secreted by actively growing cells exhibits a significantly higher level of phosphorylation than does the fibronectin secreted by quiescent cells of the same species.     

Nerve Growth Factor Mediates Monosialoganglioside-Induced Release of Fibronectin and J1/Tenascin from C6 Glioma Cells

C6 rat glioma cells incubated in serum-free medium with D-[14C]glucosamine secrete, on stimulation with nerve growth factor (NGF) or monosialogangliosides (MSGs), several glycoproteins (Gps), the most prominent of which are a 270-, 220-, and 69-kDa Gp. Several growth factors, hormones, phorbol ester, and disialo- and trisialogangliosides did not stimulate secretion. Western blot analysis of the conditioned medium from C6 cells stimulated with NGF or MSG identified one distinct band of approximately 220 kDa for fibronectin and J1/tenascin, which comigrated. Antiserum to NGF prevented NGF-stimulated release and also blocked MSG-evoked release. The 220-kDa band was labeled after pulse labeling with [35S]methionine in the presence of NGF, and by a 15-min chase period radioactively labeled J1/tenascin could be immunoprecipitated. Tunicamycin drastically inhibited almost completely release of the 220-kDa Gp labeled by D-[14C]glucosamine or [35S]methionine. These results extend the range of neurotrophic properties attributed to NGF to cells of glial origin and suggest that NGF regulates secretion of extracellular matrix proteins. MSG stimulation of fibronectin and J1/tenascin secretion may be mediated by NGF or an NGF-like molecule also secreted by the C6 glioma cells.