772 research outputs found
Simian immunodeficiency virus (SIV(mac)251) membrane lipid mixing with human CD4\u3csup\u3e+\u3c/sup\u3e and CD4\u3csup\u3e-\u3c/sup\u3e cell lines in vitro does not necessarily result in internalization of the viral core proteins and productive infection
The cell binding site of simian immunodeficiency virus (SIV) is believed to be the CD4 molecule. Several CD4+ cell lines are, however, resistant to infection by SIV(mac)251 in vitro and additional cell membrane molecules have been implicated in SIV(mac)251 entry. We investigated the binding, envelope fusion and entry of the viral core proteins (p27) of SIV(mac)251 into two human CD4+ cell lines (H9 and Sup-T1) which are infectible, and one CD4+ (A3.01) and two CD4- cell lines (K562 and Raji) that are resistant to infection. The fusion of the viral and cellular membranes was monitored by a fluorescence assay for lipid mixing. Cell entry of the viral core was evaluated following virus-cell incubation and cell surface trypsinization. We found that SIV(mac)251 can bind to and fuse (membrane lipid mixing) in a temperature dependent but pH-independent fashion with CD4+ and CD4- human-derived cell lines. In contrast. lipid mixing with CD4 expressing EL-4 mouse T cells or Mv-1-lu mink lung fibroblasts was absent or limited, suggesting that certain components of human cell membranes in addition to CD4 are involved in SIV(mac) envelope-cell fusion. Lipid mixing with the human cells was inhibited partially by soluble CD4. Anti-CD4 antibodies inhibited the lipid inter-mixing with H9, but not with Raji cells, whereas neutralizing anti-SIV(mac) sera inhibited fusion with both CD4+ and CD4- cells. Out of the five human cell lines tested, efficient entry of p27 and productive infection took place only with H9 and Sup-T1 cells. In these two cases, the amounts of p27 internalized during virus-cell fusion correlated with the extent of infection
Eradication of human immunodeficiency virus type-1 (HIV-1)-infected cells
Predictions made soon after the introduction of human immunodeficiency virus type-1 (HIV-1) protease inhibitors about potentially eradicating the cellular reservoirs of HIV-1 in infected individuals were too optimistic. The ability of the HIV-1 genome to remain in the chromosomes of resting CD4+ T cells and macrophages without being expressed (HIV-1 latency) has prompted studies to activate the cells in the hopes that the immune system can recognize and clear these cells. The absence of natural clearance of latently infected cells has led to the recognition that additional interventions are necessary. Here, we review the potential of utilizing suicide gene therapy to kill infected cells, excising the chromosome-integrated HIV-1 DNA, and targeting cytotoxic liposomes to latency-reversed HIV-1-infected cells
Bacteriophage therapy of bacterial infections: The rediscovered frontier
Antibiotic-resistant infections present a serious health concern worldwide. It is estimated that there are 2.8 million antibiotic-resistant infections and 35,000 deaths in the United States every year. Such microorganisms include Acinetobacter, Enterobacterioceae, Pseudomonas, Staphylococcus and Mycobacterium. Alternative treatment methods are, thus, necessary to treat such infections. Bacteriophages are viruses of bacteria. In a lytic infection, the newly formed phage particles lyse the bacterium and continue to infect other bacteria. In the early 20th century, d’Herelle, Bruynoghe and Maisin used bacterium-specific phages to treat bacterial infections. Bacteriophages are being identified, purified and developed as pharmaceutically acceptable macromolecular “drugs,” undergoing strict quality control. Phages can be applied topically or delivered by inhalation, orally or parenterally. Some of the major drug-resistant infections that are potential targets of pharmaceutically prepared phages are Pseudomonas aeruginosa, Mycobacterium tuberculosis and Acinetobacter baumannii
Efficient gene transfer by transferrin lipoplexes in the presence of serum
Cationic lipids are being used increasingly as reagents for gene delivery both in vitro and in vivo. One of the limitations to the application of cationic lipid-DNA complexes (lipoplexes) in vivo is the inhibition of gene delivery by serum. In this study, we have shown that transferrin (Tf)-lipoplexes, which had transferrin adsorbed at their surface via electrostatic interactions, are much more effective than plain lipoplexes in transfecting cells in the presence of relatively high concentrations (up to 60%) of fetal bovine serum (FBS). Serum even enhanced transfection by Tf-lipoplexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/dioleoylphosphatidylethanolamine (DOPE)/pCMVLacZ at high lipid/DNA (+/-) charge ratios, and inhibited lipofection for those with low charge ratios when they were added to the cells immediately after the preparation of complexes. The effect of serum on lipofection was dose-dependent. Preincubation of the complexes at 20°C for 6 h led to serum resistance, even for the negatively charged transferrin-lipoplexes. A similar tendency was observed for DOTAP/cholesterol and DOTAP/DOPE/cholesterol liposomes. The percentage of cells transfected, measured by β-galactosidase expression, also increased with the serum concentration. Cell viability was not affected significantly when the cells were incubated with the complexes for 4 h at 37°C, followed by a 48-h incubation. Our findings extend the scope of previous studies where transferrin-lipoplexes were used to introduce DNA into cells, rendering these complexes and their future derivatives potential alternatives to viral vectors for gene delivery in vivo. Copyright (C) 2000 Elsevier Science B.V
The morphometric, meristic and genetic characteristics of European sprat (Sprattus sprattus) in the Black Sea
The European sprat (Sprattus sprattus) represents the unit stock shared among the Black Sea countries. It has a key role in the Black Sea ecosystem and is determined by its importance from both a commercial and an ecological point of view. Up to now, there are few studies on the meristic, morphometric and genetic characteristics of this species. In this study, the species were specified and studied for meristic, morphometric and genetic characteristics. Mitochondrial gene regions which 16S rRNA and cytochrome c oxidase subunit I (COI), were studied and compared with some of the family Clupeidae species in the National Center for Biotechnology Information (NCBI) and the phylogenetic relationship was examined. A total of 14 haplotypes were determined for the mitochondrial COI gene region and 3 haplotypes were determined for the 16S rRNA gene region. This study will contribute to the sustainable management of this species as well as be a basis for future studies
Synexin facilitates fusion of specific phospholipid membranes at divalent cation concentrations found intracellularly.
The effect of synexin (an adrenal medullary protein) on the kinetics of Ca2+- and Mg2+-mediated membrane fusion was examined. Membrane fusion was studied by monitoring intermixing of the aqueous contents of phospholipid vesicles. Synexin facilitated Ca2+-mediated, but not Mg2+-mediated, fusion of phosphatidate/phosphatidylethanolamine (1:3) and phosphatidate/phosphatidylserine/phosphatidylethanolamine/cholesterol (1:2:3:2) vesicles. The threshold concentration of Ca2+ for fusion was decreased to approximately equal to 10 microM in the presence of synexin at 6 micrograms/ml and 1.5 mM Mg2+ in vesicle suspensions containing 50 microM lipid. This effect of synexin was drastically inhibited by including 25% phosphatidylcholine (mol/mol) in the vesicle membrane. It is proposed that the Ca2+-dependent lipid-specific enhancement of membrane fusion by synexin contributes to an increase in the sensitivity of specific intracellular membranes to Ca2+ with respect to fusion
Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies
Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy.status: publishe
A monoclonal antibody to the gp120-CD4 complex has differential effects on HIV-induced syncytium formation and viral infectivity
A murine monoclonal antibody (MAb F-91-55) raised against the complex of soluble CD4 and human immunodeficiency virus type 1 (HIV-1) gp120 had previously been found to inhibit syncytium formation without inhibiting the interaction of CD4 with gp120, and its binding site was localized within the first two domains (D1/D2) of CD4. We investigated whether this antibody inhibited the infectivity of HIV-1 in the CD4+ T cell lines A3.01, Sup-T1 and H9. We also examined the effect of the antibody on syncytium formation between these cells and chronically infected H9 cells. Syncytium formation was found to depend critically on the incubation medium used. The effect of the MAb on HIV-1 infectivity was very limited with A3.01 and Sup-T1 cells, although it inhibited syncytium formation between A3.01 or Sup-T1 and chronically infected H9 cells. In contrast, the MAb inhibited significantly the infectivity of HIV-1 in H9 cells, but it also inhibited syncytium formation between H9 and chronically infected H9 cells to a greater extent than in the case of the other cell lines. Our results indicate that cellular systems used for syncytium assays differ in their susceptibility to inhibitory antibodies. In the A3.01 and Sup-T1 cell systems, the differences in the ability of the MAb to block viral entry or syncytium formation raise the possibility that the mechanisms of interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion
Human immunodeficiency virus type-1 (HIV-1) infection increases the sensitivity of macrophages and THP-1 cells to cytotoxicity by cationic liposomes
Cationic liposomes may be valuable for the delivery of anti-sense oligonucleotides, ribozymes, and therapeutic genes into human immunodeficiency virus type 1 (HIV-1)-infected and uninfected cells. We evaluated the toxicity of three cationic liposomal preparations, Lipofectamine, Lipofectin, and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE) reagent, to HIV-infected and uninfected cells. Monocyte/macrophages were infected with HTV-1(BaL) and treated with liposomes in medium containing 20% fetal bovine serum (FBS) for 4 h or 24 h at 37°C. Uninfected monocytic THP-1 cells and chronically infected THP-1/HIV-1(IIIB) cells were treated with phorbol 12-myristate 13-acetate (PMA) and exposed to liposomes in the presence of 10% FBS. Toxicity was evaluated by the Alamar Blue assay and viral p24 production. The toxic effect of cationic liposomes was very limited with uninfected cells, although concentrations of liposomes that were not toxic within a few days of treatment could cause toxicity at later times. In HIV-1(BaL)-infected macrophages, Lipofectamine (up to 8 μM) and Lipofectin (up to 40 μM) were not toxic after a 4-h treatment, while DMRIE reagent at 40 μM was toxic. While a 4-h treatment of THP-1/HIV-1(IIIB) cells with the cationic liposomes was not toxic, even up to 14 days post-treatment, all three cationic liposomes were toxic to cells at the highest concentration tested after a 24-h treatment. Similar results were obtained with the Alamar Blue assay, Trypan Blue exclusion and a method that enumerates nuclei. Infected cells with relatively high overall viability could be impaired in their ability to produce virions, indicating that virus production appears to be more sensitive to treatment with the cationic liposomes than cell viability. Our results indicate that HIV-infected cells are more susceptible than uninfected cells to killing by cationic liposomes. The molecular basis of this differential effect is unknown; it is proposed that alterations in cellular membranes during virus budding cause enhanced interactions between cationic liposomes and cellular membranes
Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion
We have examined the early events in Ca2+-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol. © 1982
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