Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts

Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.     

Cell fusion induced by lysolecithin and concanavalin A in Drosophila melanogaster somatic cells cultured in vitro

Cell fusion and homokaryon formation were induced with lysolecithin and concanavalin A (ConA) in two karyotypically different embryonic cell lines of Dr. melanogaster, GM 1 and IB 5. The fusion capacity and the cytotoxic activity of LL, analysed immediately after treatment, were more rapid and drastic than those of ConA. Moreover, the two lines considered consistently differed in their sensitivity to the chemical agents: GM 1 cells were more sensitive and less efficient in fusion than IB 5 cells.     

Annexin A1: novel roles in skeletal muscle biology

Annexin A1 (ANXA1, lipocortin-1) is the first characterized member of the annexin superfamily of proteins, so called since their main property is to bind (i.e., to annex) to cellular membranes in a Ca(2+) -dependent manner. ANXA1 has been involved in a broad range of molecular and cellular processes, including anti-inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis, and differentiation. New insights show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. This suggests that ANXA1 may contribute to the regeneration of skeletal muscle tissue and may have therapeutic implications with respect to the development of ANXA1 mimetics.     

Probabilistic finite element prediction of knee wear simulator mechanics

Computational models have recently been developed to replicate experimental conditions present in the Stanmore knee wear simulator. These finite element (FE) models, which provide a virtual platform to evaluate total knee replacement (TKR) mechanics, were validated through comparisons with experimental data for a specific implant. As with any experiment, a small amount of variability is inherently present in component alignment, loading, and environmental conditions, but this variability has not been previously incorporated in the computational models. The objectives of the current research were to assess the impact of experimental variability on predicted TKR mechanics by determining the potential envelope of joint kinematics and contact mechanics present during wear simulator loading, and to evaluate the sensitivity of the joint mechanics to the experimental parameters. In this study, 8 component alignment and 4 experimental parameters were represented as distributions and used with probabilistic methods to assess the response of the system, including interaction effects. The probabilistic FE model evaluated two levels of parameter variability (with standard deviations of component alignment parameters up to 0.5mm and 1 degrees ) and predicted a variability of up to 226% (3.44mm) in resulting anterior-posterior (AP) translation, up to 169% (4.30 degrees ) in internal-external (IE) rotation, but less than 10% (1.66MPa) in peak contact pressure. The critical alignment parameters were the tilt of the tibial insert and the IE rotational alignment of the femoral component. The observed variability in kinematics and, to a lesser extent, contact pressure, has the potential to impact wear observed experimentally.     

Immunodetection of the microvillous cytoskeleton molecules villin and ezrin in the parasitophorous vacuole wall of Cryptosporidium parvum (Protozoa: Apicomplexa)

Microvilli - actin - villin - ezrin - Cryptosporidium parvum The sporozoites and merozoites of the Apicomplexan protozoan Cryptosporidium parvum (C. parvum) invade the apical side of enterocytes and induce the formation of a parasitophorous vacuole which stays in the brush border area and disturbs the distribution of microvilli. The vacuole is separated from the apical cytoplasm of the cell by an electron-dense layer of undetermined composition. In order to characterize the enterocyte cytoskeleton changes that occur during C. parvum invasion and development, we used both confocal immunofluorescence and immunoelectron microscopy to examine at the C.parvum-enterocyte interface the distribution of three components of the microvillous skeleton, actin, villin and ezrin. In infected cells, rhodamine-phalloidin and anti-villin and anti-ezrin antibodies recognized ring-like structures surrounding the developing parasites. By immunoelectron microscopy, both villin and ezrin were detected in the parasitophorous vacuole wall surrounding the luminal and lateral sides of the intracellular parasite. In contrast, anti-beta and anti-gamma actin antibodies showed no significant labelling of the vacuolar wall. These observations indicate that the parasitophorous vacuole wall contains at least two microvillus-derived components, villin and ezrin, as well as a low amount of F-actin. These data suggest that C.parvum infection induces a rearrangement of cytoskeleton molecules at the apical pole of the host cell that are used to build the parasitophorous vacuole.     

The role of carbon-donor hydrogen bonds in stabilizing tryptophan conformations

Although most side-chain torsion angles correspond to low-energy rotameric positions, deviations occur with significant frequency. One striking example arises in Trp residues, which have an important role in stabilizing protein structures because of their size and mixed hydrophobic/hydrophilic character. Ten percent of Trp side-chains have unexplained conformations with chi(2) near 0 degrees instead of the expected 90 degrees. The current work is a structural and energetic analysis of these conformations. It is shown that many Trp residues with these orientations are stabilized by three-center carbon-donor hydrogen bonds of the form C-H...X...H-C, where X is a polar hydrogen-bond acceptor in the environment of the side-chain. The bridging hydrogen bonds occur both within the Trp side-chain and between the side-chain and the local protein backbone. Free energy maps of an isolated Trp residue in an explicit water environment show a minimum corresponding to the off-rotamer peak observed in the crystallographic data. Bridging carbon-donor hydrogen bonds are also shown to stabilize on-rotamer Trp conformations, and similar bridging hydrogen bonds also stabilize some off-rotamer Asp conformations. The present results suggest a previously unrecognized role for three-center carbon-donor hydrogen bonds in protein structures and support the view that the off-rotamer Trp side-chain orientations are real rather than artifacts of crystallographic refinements. Certain of the off-rotamer Trp conformations appear to have a functional role.