Quadruplex structure of Oxytricha telomeric DNA oligonucleotides.

The telomeres of most eukaryotes contain a repeating G-rich sequence with the consensus d(T/A)1-4G1-8, of which 12-16 bases form a 3' single-strand overhang beyond the telomeric duplex. It has been proposed that these G-rich oligonucleotides associate to form four-stranded structures from one, two or four individual strands and that these structures may be relevant in vivo. The proposed structures contain Hoogsteen base-paired G-quartets, precedent for which has been in the literature for many years. Here we use 1H NMR spectroscopy to study the conformations of the DNA oligonucleotides d(G4T4G4) (Oxy-1.5) and d(G4T4G4T4G4T4G4) (Oxy-3.5) which contain the Oxytricha telomere repeat (T4G4). We find that these molecules fold to form a symmetrical bimolecular and an intramolecular quadruplex, respectively. Both structures have four G-quartets formed from nucleotides that are alternately syn and anti along each strand. This arrangement differs from earlier models in which the strands are alternately all syn or all anti. The T4 loops in Oxy-1.5 are on opposite ends of the quadruplex and loop diagonally across the G-quartet, resulting in adjacent strands being alternately parallel and antiparallel.     

Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.     

Rapid switching to multiple antigenic and adhesive phenotypes in malaria.

Adhesion of parasitized erythrocytes to post-capillary venular endothelium or uninfected red cells is strongly implicated in the pathogenesis of severe Plasmodium falciparum malaria. Neoantigens at the infected red-cell surface adhere to a variety of host receptors, demonstrate serological diversity in field isolates and may also be a target of the host-protective immune response. Here we use sequential cloning of P. falciparum by micromanipulation to investigate the ability of a parasite to switch antigenic and cytoadherence phenotypes. Our data show that antigens at the parasitized cell surface undergo clonal variation in vitro in the absence of immune pressure at the rate of 2% per generation with concomitant modulations of the adhesive phenotype. A clone has the potential to switch at high frequency to a variety of antigenic and adhesive phenotypes, including a new type of cytoadherence behaviour, 'auto-agglutination' of infected erythrocytes. This rapid appearance of antigenic and functional heterogeneity has important implications for pathogenesis and acquired immunity.     

Superantigen implicated in dependence of HIV-1 replication in T cells on TCR V beta expression.

In the pathogenesis of AIDS it is not yet understood whether the small fraction of CD4+ T cells (approximately 1%) infected with the human immunodeficiency virus (HIV) are randomly targeted or not. Here we present evidence that human CD4 T-cell lines expressing selected T-cell antigen receptor V beta gene products can all be infected in vitro with HIV-1, but give markedly different titres of HIV-1 virion production. For example, V beta 12 T-cell lines from several unrelated donors reproducibly yielded up to 100-fold more gag gene product (p24gag antigen) than V beta 6.7a lines. This is consistent with a superantigen effect, because the V beta selectivity was observed with several divergent HIV-1 isolates, was dependent on antigen-presenting cells and on major histocompatibility complex (MHC) class II but was not MHC class II-restricted. The in vivo significance of these findings is supported by the preferential stimulation of V beta 12+ T cells by freshly obtained irradiated antigen-presenting cells from some HIV-1-seropositive but not HIV-1-negative donors. Moreover, cells from patients positive for viral antigen (gp120) were enriched in the V beta 12 subpopulation. V beta 12+ T cells were not deleted in AIDS patients, however, raising the possibility that a variety of mechanisms contribute to T-cell depletion. Our results indicate that a superantigen targets a subpopulation of CD4+ cells for viral replication.     

Cytotoxic T-lymphocyte activation involves a cascade of signalling and adhesion events.

In addition to the antigen-specific T-cell receptor (TCR), T cells bear an array of 'accessory' molecules that can contribute to stable adhesion to the antigen-bearing cell and provide costimulatory signals. For several of these, T-cell adhesion to the ligand can be activated by TCR-dependent signalling (a signal from the TCR primes the coreceptor to bind to its ligand). It is unclear whether the individual coreceptors share common mechanisms of priming and cosignalling, and perhaps act in a redundant manner, or whether they act in a distinct way and contribute uniquely to the activation process. We report here the use of isolated alloantigen, class I proteins and fibronectin ligands to show that coreceptors on cytotoxic T lymphocytes are activated sequentially and deliver distinct biochemical signals on binding to their ligands. TCR engagement activates CD8 by a protein tyrosine kinase-dependent pathway, and CD8 then acts as a signal for initiation of polyphosphoinositide hydrolysis on binding to class I. In contrast, activated adhesion to fibronectin does not initiate polyphosphoinositide hydrolysis, but amplifies hydrolysis once it has been initiated. Thus, cytotoxic T-lymphocyte activation involves a TCR-initiated cascade of adhesion and signalling events leading to response.     

Human infection by genetically diverse SIVSM-related HIV-2 in west Africa.

Our understanding of the biology and origins of human immunodeficiency virus type 2 (HIV-2) derives from studies of cultured isolates from urban populations experiencing epidemic infection and disease. To test the hypothesis that such isolates might represent only a subset of a larger, genetically more diverse group of viruses, we used nested polymerase chain reactions to characterize HIV-2 sequences in uncultured mononuclear blood cells of two healthy Liberian agricultural workers, from whom virus isolation was repeatedly unsuccessful, and from a culture-positive symptomatic urban dweller. Analysis of pol, env and long terminal repeat regions revealed the presence of three highly divergent HIV-2 strains, one of which (from one of the healthy subjects) was significantly more closely related to simian immunodeficiency viruses infecting sooty mangabeys and rhesus macaques (SIVSM/SIVMAC) than to any virus of human derivation. This subject also harboured multiply defective viral genotypes that resulted from hypermutation of G to A bases. Our results indicate that HIV-2, SIVSM and SIVMAC comprise a single, highly diverse group of lentiviruses which cannot be separated into distinct phylogenetic lineages according to species of origin.     

S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28.

In somatic cells, entry into mitosis depends on the completion of DNA synthesis. This dependency is established by S-phase feedback controls that arrest cell division when damaged or unreplicated DNA is present. In the fission yeast Schizosaccharomyces pombe, mutations that interfere with the phosphorylation of tyrosine 15 (Y15) of p34cdc2, the protein kinase subunit of maturation promoting factor, accelerate the entry into mitosis and abolish the ability of unreplicated DNA to arrest cells in G2. Because the tyrosine phosphorylation of p34cdc2 is conserved in S. pombe, Xenopus, chicken and human cells, the regulation of p34cdc2-Y15 phosphorylation could be a universal mechanism mediating the S-phase feedback control and regulating the initiation of mitosis. We have investigated these phenomena in the budding yeast Saccharomyces cerevisiae. We report here that the CDC28 gene product (the S. cerevisiae homologue of cdc2) is phosphorylated on the equivalent tyrosine (Y19) during S phase but that mutations that prevent tyrosine phosphorylation do not lead to premature mitosis and do not abolish feedback controls. We have therefore demonstrated a mechanism that does not involve tyrosine phosphorylation of p34 by which cells arrest their division in response to the presence of unreplicated or damaged DNA. We speculate that this mechanism may not involve the inactivation of p34 catalytic activity.     

Threshold and suprathreshold responses of the auditory receptors in an arctiid moth

Considering the responses of both auditory receptors inEmpyreuma pugione (Arctiidae, Ctenuchinae), it was found that this auditory organ maintains its frequency tuning and directionality over an intensity range of more than 60 dB. For the first time in the Lepidoptera we demonstrate a close frequency match between auditory sensitivity and the power spectrum of the sound emitted by the same species.