Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.     

Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages

Glycosylphosphatidylinositol (GPI)-anchored proteins are involved in cell wall integrity and cell-cell interactions. We disrupted the Candida albicans homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a GPI anchor-modifying activity. In the mutant and on solid media, the yeast-to-hyphae transition was blocked, whereas chlamydospore formation was enhanced. However, the morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis and cell shape were observed in both liquid and solid media. The cell wall structure was also modified in the mutants, as shown by hypersensitivity to Calcofluor white. In vitro and in vivo assays revealed that the mutant interacted with its host in a modified way, resulting in reduced virulence in mice and reduced survival in the gastrointestinal environment of mice. The mitogen-activated protein (MAP) kinase pathway of macrophages was downregulated by the wild-type cells but not by the DeltaCagpi7 null strains. In agreement with this abnormal behaviour, mutant cells were more sensitive to the lytic action of macrophages. Our results indicate that a functional GPI anchor is required for full hyphal formation in C. albicans, and that perturbation of the GPI biosynthesis results in hypersensitivity to host defences.     

Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

Background  

We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency.     

Methacrylate derivatives incorporating pyroglutamic acid

Methacrylates containing pyroglutamic acid were synthesized in good yields. Methyl alpha-pyroglutamyl methylacrylate (PyMM) and methyl alpha-pyroglutamidoundecanoyl methylacrylate (PyUM) give very fast photopolymerization rates both in homopolymerizations and with widely used commercial monomers N-vinyl pyrrolidinone (NVP) and hydroxyethyl methacrylate (HEMA). Soluble or cross-linked homopolymers can be obtained depending upon polymerization temperature. Pyroglutamic methacrylates polymerize without added initiator in the melt. Solution cast, photocured, and thermally cured coatings gave good to excellent adhesion to poly(ethylene terephthalate) and glass surfaces.     

Identification and expression of Ima,a novel Ral-interacting Drosophila protein

We report the identification of Ima, a novel Drosophila MAGUK-like protein, which contains two WW and four PDZ protein interaction domains and interacts with the small GTPase dRal in the yeast two-hybrid system and pull-down assays. The gene is expressed in distinct spatiotemporal patterns throughout embryonic development. Overexpression of Ima interferes with normal Drosophila development, indicating that the gene functions in a tissue specific manner.     

Inhibition of tumor growth in mice with severe combined immunodeficiency is mediated by heat shock protein 70 (Hsp70)-peptide-activated,CD94 positive natural killer cells

Previously, we reported that the major stress-inducible heat shock protein 70 (Hsp70) acts as a recognition structure for natural killer (NK) cells, if localized on the cell surface of tumor cells. Incubation of purified NK cells with low-dose interleukin (IL)-2 (100 IU/mL) plus recombinant Hsp70-protein or the immunogenic 14-mer Hsp70-peptide TKDNNLLGRFELSG450-463, termed TKD (2 microg/mL), enhances the cytolytic activity against Hsp70 membrane-positive (CX+) but not against Hsp70-negative (CX-) tumor cells. Here, we show that the cytolytic activity against Hsp70-positive tumor cells is inducible by incubation of unseparated peripheral blood mononuclear cells (PBMNC) with low-dose IL-2 plus TKD. Cell sorting experiments revealed that within the PBMNC population CD94(+)/CD3(-) NK cells, and not CD94(-)/CD3(+) T cells, mediate the cytotoxic activity against Hsp70-positive tumor cells. The antitumoral effect of PBMNC stimulated either with IL-2 plus TKD or with IL-2 alone was assessed in tumor-bearing severe combined immunodeficiency/beige mice. A single intravenous (iv) injection of 40 x 10(6) IL-2 plus TKD-stimulated PBMNC (containing 5.2 x 10(6) NK cells) on day 4 results in a 60% reduction in tumor size, from 3.89 g to 1.56 g. In contrast, the adoptive transfer of the identical amount PBMNC stimulated with low-dose IL-2 only (containing 4.4 x 10(8) NK cells) reduces the tumor size only less than 10% (3.64 g). A phenotypic characterization of the excised tumors revealed that predominantly Hsp70-positive tumor cells were eliminated by TKD-activated PBMNC. Kinetic studies demonstrate that the in vivo cytolytic capacity of TKD-stimulated PBMNC is dependent on the effector to target cell ratio. An iv injection of effector cells on day 1 or 2 after tumor cell inoculation results in significantly smaller tumors (0.77 g or 0.89 g) on day 21 as compared with mice that were immunoreconstituted on day 4 or 8 (1.39 g or 2.23 g). The tumor size of nonimmunoreconstituted control animals was 3.55 g.