Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.     

Systemic administration of cationic phosphonolipids/DNA complexes and the relationship between formulation and lung transfection efficiency

Performances of cationic lipid formulations for intravenous gene delivery to mouse lungs have been previously reported. We report in this study that cationic phosphonolipids, when appropriately formulated, can be good synthetic vectors for gene delivery to lung after intravenous administration. One of our reagents, GLB43, was capable of mediating a 500-fold higher expression in the lungs of mice than could be obtained with free pDNA alone (P=0.018). We demonstrate that the most important parameters for cationic phosphonolipid transfection activity after systemic administration are the chemical structure of the cationic phosphonolipid, the lipid to DNA charge ratio and the inclusion of co-lipid in the formulation. We report using a luciferase reporter gene that transfection activity in vivo 24 h after cationic phosphonolipid systemic administration could not be predicted from in vitro analysis. In contrast to in vitro studies, cationic phosphonolipids including the oleyl acyl chains (GLB43) were more effective than its analogue with the myristyl acyl chains (GLB73). Using pathological analysis of animal livers, we demonstrate that the toxicity level was correlated with the lipoplex formulation and the lipid to DNA ratio.     

Stimulation of rat cutaneous fibroblasts and their synthetic activity by implants of powdered nacre (mother of pearl)

The components of the cutaneous envelope, the epidermis and the dermis, change in response to aging or environmental stress factors. The fibroblasts involved in maintaining skin tone are the main targets. Nacre, mother of pearl, from Pinctada maxima, which can stimulate and regulate bone forming cells, was implanted in the dermis of rats to test its action on the skin fibroblasts. This report describes the effect of nacre on the skin fibroblast recruitment and physiological activity. It resulted in enhanced extracellular matrix synthesis and the production of components implicated in cell to cell adhesion and communication (such as decorine) and in tissue regeneration (type I and type III collagens). The nacre implant produced a well vascularized tissue. The physiological conditions in the region around the implant are thus those required for the positive interactions between the dermis and epidermis which are fundamental for the physiological function of the skin.     

Modelling T-cell-mediated suppression dependent on interactions in multicellular conjugates

Tolerance to peripheral body antigens involves multiple mechanisms, namely T-cell-mediated suppression of potentially autoimmune cells. Recent in vivo and in vitro evidence indicates that regulatory T cells suppress the response of effector T cells by a mechanism that requires the simultaneous conjugation of regulatory and effector T cells with the same antigen-presenting cell (APC). Despite this strong requirement, it is not yet clear what happens while both cells are conjugated. Several hypotheses are discussed in the literature. Suppression may result from simple competition of regulatory and effector cells for activation resources on the APC; regulatory T cells may deliver an inhibitory signal to effector T cells in the same conjugate; or effector T cells may acquire the regulatory phenotype during their interaction with regulatory T cells. The present article tries to further our understanding of T-cell-mediated suppression, and to narrow-down the number of candidate mechanisms. We propose the first general formalism describing the formation of multicellular conjugates of T cells and APCs. Using this formalism we derive three particular models, representing alternative mechanisms of T-cell-mediated suppression. For each model, we make phase plane and bifurcation analysis, and identify their pros and cons in terms of the relationship with the large body of experimental observations on T-cell-mediated suppression. We argue that accounting for the quantitative details of adoptive transfers of tolerance requires models with bistable regimes in which either regulatory cells or effectors cells dominate the steady state. From this analysis, we conclude that the most plausible mechanism of T-cell-mediated suppression requires that regulatory T cells actively inhibit the growth of effector T cells, and that the maintenance of the population of regulatory T cells is dependent on the effector T cells. The regulatory T cell population may depend on a growth factor produced by effector T cells and/or on a continuous differentiation of effector cells to the regulatory phenotype.     

Phylogenetic Relationships of Acanthocephala Based on Analysis of 18S Ribosomal RNA Gene Sequences

Acanthocephala (thorny-headed worms) is a phylum of endoparasites of vertebrates and arthropods, included among the most phylogenetically basal tripoblastic pseudocoelomates. The phylum is divided into three classes: Archiacanthocephala, Palaeacanthocephala, and Eoacanthocephala. These classes are distinguished by morphological characters such as location of lacunar canals, persistence of ligament sacs in females, number and type of cement glands in males, number and size of proboscis hooks, host taxonomy, and ecology. To understand better the phylogenetic relationships within Acanthocephala, and between Acanthocephala and Rotifera, we sequenced the nearly complete 18S rRNA genes of nine species from the three classes of Acanthocephala and four species of Rotifera from the classes Bdelloidea and Monogononta. Phylogenetic relationships were inferred by maximum-likelihood analyses of these new sequences and others previously determined. The analyses showed that Acanthocephala is the sister group to a clade including Eoacanthocephala and Palaeacanthocephala. Archiacanthocephala exhibited a slower rate of evolution at the nucleotide level, as evidenced by shorter branch lengths for the group. We found statistically significant support for the monophyly of Rotifera, represented in our analysis by species from the clade Eurotatoria, which includes the classes Bdelloidea and Monogononta. Eurotatoria also appears as the sister group to Acanthocephala. Received: 12 October 1999 / Accepted: 8 February 2000     

Mutation of a conserved residue (D123) required for oligomerization of human immunodeficiency virus type 1 Nef protein abolishes interaction with human thioesterase and results in impairment of Nef biological functions

Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.     

Distinct trafficking pathways mediate Nef-induced and clathrin-dependent major histocompatibility complex class I down-regulation

The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.