Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine⅐oligopurine sequences and therefore are candidate molecules for artificial gene regulation, in vitro and in vivo. We recently have described oligonucleotide analogues containing N3-P5 phosphoramidate (np) linkages that exhibited efficient inhibition of transcription elongation in vitro. In the present work we provide conclusive evidence that np-modified TFOs targeted to the HIV-1 polypurine tract (PPT) sequence can inhibit transcriptional elongation in cells, either in transient or stable expression systems. The same constructs were used in transient expression assays (target sequence on transfected plasmid) and in the generation of stable cell lines (target sequence integrated into cellular chromosomes). In both cases the only distinguishable feature between the cellular systems is the presence of an insert containing the wild-type PPT͞HIV-1 sequence, a mutated version with two mismatches, or the absence of the insert altogether. The inhibitory action induced by np-TFOs was restricted to the cellular systems containing the complementary wild-type PPT͞HIV-1 target, and consequently can be attributed only to a triple-helix-mediated mechanism. As a part of this study we also have applied an imaging technique to quantitatively investigate the dynamics of TFO-mediated specific gene silencing in single cells.

Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Bradykinin (BK)1 and kallidin are biological active peptides generated by the proteolytic cleavage of kininogens by serine proteases of the kallikrein family. High molecular weight kininogens are precursors of BK, whereas low molecular weight kininogens give origin to kallidin. Along with other kinins, BK and kallidin elicit a wide range of physiological responses, being classically involved in the control of cardiovascular homeostasis and inflammation. As a matter of fact, altered function of the kallikrein-kinin system has been implicated in the development of various pathological conditions such as arthritis, pancreatitis, and asthma (for a review see Refs. 1 and 2). Emerging evidence shows that kinins are stored in neuronal cells of the central nervous system and may act as neuromediators in various functions, including the control of nociceptive information (for a review see Ref.3). Expression of kallikrein in developing rat brains (4) supports the notion that kinin-induced receptor activity might be required during neuronal development. BK has also been shown to enhance the release of neurotransmitters such as noradrenalin and neuropeptide Y by sympathetic neurons, chromaffin cells, and pheochromocytoma cells (5-8). Moreover, BK implication in the control of calcium homeostasis has already been demonstrated in adult sensory neurons (9).Most of the biological actions of BK and kallidin are mediated by a serpentine receptor coupled to a G-protein, the kinin-B2 receptor (B2BKR), which is constitutively expressed and widely distributed throughout central and peripheral tissues under physiological conditions. However, there is evidence that expression of B2BKRs during development is regulated. For instance, B2BKR expression has been shown to be involved in the development of the urinary and cardiovascular systems (10). Inhibition of B2BKR activity in rat embryos resulted in animals with disturbed kidney development (11). Besides being regulated during the ontogenesis of cardiovascular and urinary systems, a large set of evidence exists showing that modulation of B2 receptor expression and function also appears during neuronal development. Thus, it has been detected in central and peripheral noradrenergic neurons, in the spinal cord, in neuronal differentiating PC12 pheochromocytoma cells, and in neuroblastoma and glia-derived cell lines (12-18). A cross-talk between the B2BKR and other hormone and neurotransmitter

SUMMARYArteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Deltalike 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic lossand gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4 +/− mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4 +/− mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

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The ability to specifically manipulate gene expression has wide-ranging applications in experimental biology and in gene-based therapeutics. The design of molecules that recognise specific sequences on the DNA double helix provides us with interesting tools to interfere with DNA information processing at an early stage of gene expression. Triplex-forming molecules specifically recognise oligopyrimidine-oligopurine sequences by hydrogen bonding interactions. Applications of such triplex-forming molecules (TFMs) are the subject of the present review. In cell cultures, TFMs have been successfully used to down- or up-regulate transcription in a gene-specific manner and to induce genomic DNA modifications at a selected site. The first evidence of a triplex-based activity in animals has been provided recently. In addition, TFMs are also powerful tools for gene-specific chemistry, in particular for gene transfer applications.

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K409A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F1 mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K409A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K409A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.

Chromosome localization in the interphase nuclei of eukaryotes depends on gene replication and transcription. Little is known about chromosome localization in protozoan parasites such as trypanosomes, which have unique mechanisms for the control of gene expression, with most genes being posttranscriptionally regulated. In the present study, we examined where the chromosomes are replicated in Trypanosoma cruzi, the agent of Chagas' disease. The replication sites, identified by the incorporation of 5-bromodeoxyuridine, are located at the nuclear periphery in proliferating epimastigote forms in the early S phase of the cell cycle. When the S phase ends and cells progress through the cell cycle, 5-bromodeoxyuridine labeling is observed in the nuclear interior, suggesting that chromosomes move. We next monitored chromosome locations in different stages of the cell cycle by using a satellite DNA sequence as a probe in a fluorescence in situ hybridization assay. We found two distinct labeling patterns according to the cell cycle stage. The first one is seen in the G 1 phase, in hydroxyurea-arrested epimastigotes or in trypomastigotes, which are differentiated nondividing forms. In all of these forms the satellite DNA is found in dots randomly dispersed in the nucleus. The other pattern is found in cells from the S phase to the G 2 phase. In these cells, the satellite DNA is found preferentially at the nuclear periphery. The labeling at the nuclear periphery disappears only after mitosis. Also, DNA detected with terminal deoxynucleotidyl transferase is found distributed throughout the nuclear space in the G 1 phase but concentrated at the nuclear periphery in the S phase to the G 2 phase. These results strongly suggest that T. cruzi chromosomes move and, after entering the S phase, become constrained at the nuclear periphery, where replication occurs.The eukaryotic nucleus has functional compartments for DNA replication and transcription and for RNA processing (6, 24, 36). Nuclear integrity is important for DNA synthesis initiation and for replication fork formation (7), and disruption of nuclear lamina organization prevents the elongation phase of DNA replication (29). Chromosome localization in the mammalian cell nucleus is also determined by the transcriptional status of genes. Actively transcribed genes are located in the nuclear interior, replicating early in the S phase of the cell cycle, while repressed genes, which correspond to heterochromatic regions, replicate later and are located at the nuclear periphery and around the nucleolus (11,28,32).Visualization of replication sites by labeling of cells with 5-bromodeoxyuridine (BrdU) or biotinylated dUTP (31) during the cell cycle revealed immobile foci probably linked to a nuclear matrix. Over the last 10 to 12 years, detailed information on the organization and dynamics of interphase chromosomes has emerged; most of them have been found associated with repeated domains, such as telomeres and centromeres (10), and related to the cell cycle (16,26). More recently, ...

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