Binding of chemically-modified oligonucleotides to the double-stranded stem of an RNA hairpin.

We monitored the binding of triplex-forming oligopyrimidines to the double-stranded stem of the RNA hairpin responsible for the gag-pol frameshift in HIV-1. Whereas the substitution of 5, propynyl-C for C had a limited effect, the use of a Peptide Nucleic Acid 12mer led to a drastic reduction in the stability of the oligomer/RNA complex.

Binding of oligopyrimidines to the RNA hairpin responsible for the ribosome gag-pol frameshift in HIV-1.

The 12 bp stem of the RNA hairpin responsible for the gag-pol frameshifting of the ribosomes during translation of the polycistronic HIV-1 mRNA has a pyrimidine-rich 5' strand and, consequently, a purine-rich 3' strand. Electrophoretic mobility shift assays have shown that DNA oligopyrimidines, 12 and 20 nucleotides long (but not oligopurines or G,T-containing oligomers), designed to form triplexes actually bind to the double-stranded RNA target. RNase V1 footprinting studies have confirmed the interaction between the hairpin stem and the RNA and 2'-O-methyl oligoribonucleotide analogues of the 12-mer oligodeoxypyrimidine as well as 5 propynyl,cytosine, containing the 12-mer oligodeoxypyrimidine, bind more strongly to the RNA target than the unmodified parent DNA oligomer. The complexes formed by the RNA hairpin and either the 12-mer oligodeoxypyrimidine or the 20-mer oligopyrimidine are stable at a neutral pH and in the absence of Mg2+ but blocked neither the reverse transcription nor cell-free translation of a RNA template in which the gag-pol frameshifting hairpin was inserted at the 5' end of the luciferase open reading frame.

A fluorescent base analog for probing triple helix formation.

Benzo[g]quinazoline-2,4-(1H,3H)-dione (BgQ), a fluorescent thymine analog, was incorporated into an oligopyrimidine (III) able to give rise to a triple-stranded structure by clamping a purine 11-mer (I). The formation of the I-III complex resulted in both a shift of the fluorescence emission maximum and a decreased fluorescence intensity. No such variations were observed on the formation of a Watson-Crick duplex between I and the complementary strand in which a T residue was substituted for BgQ. Therefore, the fluorescence emission of BgQ can be used to selectively monitor the formation of triple helices.

The significance of shed membrane particles during programmed cell death in vitro, and in vivo, in HIV-1 infection.

The plasma membrane remodeling, including the early transverse redistribution of phosphatidylserine, is a general feature occurring in cells in which a death program has been induced. In most cases, studies of this kind have focused mainly on cells. In this study, we report a clear correlation between the degree of apoptosis induced by a variety of agents in several types of cultured cells and the amount of shed membrane microparticles captured in the corresponding supernatants by insolubilized annexin V, a protein showing a strong affinity for phosphatidylserine. Such particles carry membrane antigens specific of the cells they stem from, and through which capture is also feasible. Homologous circulating microparticles were captured in peripheral blood from individuals with HIV-1 infection. A substantial proportion bore CD4 antigen. In some cases, CD4+ particles could be detected even in the absence of circulating CD4+ T cells, testifying to the presence of such resident cells in lymphoid tissues. These results suggest that shed membrane particles are one of the hallmarks of programmed cell death, of particular interest when the corresponding cells are hardly accessible.

N-acetylcysteine protects lymphocytes from nitrogen mustard-induced apoptosis.

The ability of the antioxidant N-acetylcysteine to prevent apoptosis induced in lymphocytes by nitrogen mustard (HN2) was investigated. HN2 caused a concentration-dependent induction of apoptosis on C3H murine spleen cells, as identified by two criteria: morphological features revealed by microscopical observations and DNA fragmentation visualized by the characteristic "ladder" pattern observed upon agarose gel electrophoresis, as well as by hypodiploid DNA-containing cells revealed by the flow cytometric analysis of propidium iodide labelled cells. The antioxidant N-acetylcysteine (NAC) was found to markedly reduce the occurrence of HN2-induced apoptosis in these cells. This protective effect will still obtained when NAC was added 30 min after HN2. In contrast, the pretreatment of spleen cells with this antioxidant did not provide any significant protection. We also showed that lymphocytes protected by NAC are still able to respond to a mitogenic stimulation. To gain some insight into the mechanisms underlying the cytoprotective action of NAC against HN2, we tested whether or not poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30), a nuclear enzyme that participates in the triggering of apoptosis induced by alkylating agents, is involved. We report that 6(5H)-phenanthridinone, a potent PARP inhibitor, did not affect the ability of NAC to prevent HN2-induced apoptosis under our experimental conditions. Thus, the exact mechanism by which NAC protects lymphocytes from HN2 cytotoxicity has yet to be determined.

Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells.

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7beta-hydroxycholesterol (7beta-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7beta-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may results from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.

Oxysterol-induced apoptosis in human monocytic cell lines.

Oxysterols constitute a large family of natural compounds, endowed with various biological activities including cholesterol regulation, immunosuppression and antitumoral potency. In the present study, we examine and compare the cytotoxic effects of two representative members of this family: 7 beta-hydroxycholesterol (7 beta-OH) and 25-hydroxycholesterol (25-OH), in two human monocytic cell lines, U-937 and HL-60. In both cell lines 7 beta-OH at 30 mu M induces cell death by apoptosis within the first hours of treatment. Under the same conditions and in contrast with results previously obtained with lymphoma cells, 25-OH is cytostatic only. It is interesting to note that the simultaneous treatment of U-937 cells by equimolar concentrations of 7 beta-OH and 25-OH leads to a considerably decreased induction of apoptosis. Such an effect is not observed with HL-60 cells. Taken together, these results indicate for the first time that: 1) oxysterols hydroxylated on the sterol nucleus are also able to induce apoptosis, 2) apoptosis can be induced by these substances in cells belonging to the myeloid lineage and 3) as far as apoptosis is concerned, a combined treatment with 7 beta-OH and 25-OH can lead to opposite effects depending on the cell type.

Immunosuppressive activities of 6(5H)-phenanthridinone, a new poly(ADP-ribose)polymerase inhibitor.

6(5H)-phenanthridinone, a recently identified poly(ADP-ribose)polymerase (PARP) inhibitor, is able, at micromolar concentrations, to inhibit concanavalin A-induced lymphocyte proliferation and to potentiate the effect of gamma radiation upon murine spleen cells. When added at the onset of a mixed lymphocyte culture, this compound strongly depresses the induction of primary allogeneic (anti-H2k) cytotoxic T-lymphocytes (CTLs). Lymphokine-activated killer (LAK) induction was also found to be impaired by the PARP inhibitor. Taken together, these results clearly indicate that PARP plays a key-role in immune reactions involving cytotoxicity and that 6(5H)-phenanthridinone could be considered as a potent immunomodulator.

Human T-cell receptor (TCR) alpha/beta + CD4-CD8- T cells express oligoclonal TCRs, share junctional motifs across TCR V beta-gene families, and phenotypically resemble memory T cells.

Most human T cells express the TCR alpha/beta and either CD4 or CD8 molecules (single positive, SP); however, small numbers lack CD4 and CD8. In inbred mice, alpha/beta CD4-CD8- (double negative, DN) T cells preferentially express certain beta variable region (V beta) families and may arise via unique developmental pathways. Increased percentages of alpha/beta DN T cells have been identified in some human and murine autoimmune and immunodeficiency diseases. However, their contribution to disease pathology or normal immunity is unknown. To study the cell surface phenotype and TCR diversity of human alpha/beta DN T cells, these cells were isolated from the peripheral blood of healthy adults. The proportion of alpha/beta DN T cells expressing molecules associated with activation (HLA-DR), previous exposure to antigen (CD45RO), and cytotoxic function (CD56, CD57, and CD11b) was increased relative to SP T cells. The TCR V beta repertoire of alpha/beta DN T cells was different from that of alpha/beta SP T cells, although most major gene families were present. For example, higher proportions of V beta 11, a minor gene family in peripheral blood leukocytes, were found in most alpha/beta DN T-cell samples. In contrast to mice, no dominant V beta family was used consistently in different human individuals. Within an individual alpha/beta DN T cells possessed an oligoclonal TCR beta repertoire with conservation of several distinct junctional amino acid motifs with one joined to three different V beta genes in two individuals, suggesting that these cells have undergone a selection process driven by a limited set of ligands. The possibility that they may represent, at least in part, originally SP T cells anergized by down-modulation of CD4 or CD8 must also be entertained. Overall, this study demonstrates that human peripheral blood alpha/beta DN T cells possess unique phenotypic and TCR beta repertoire characteristics when compared with the major alpha/beta SP T cell populations and thus may serve specialized immunologic functions and/or have an unusual origin.