Sleep deprivation before stroke is neuroprotective: a pre-ischemic conditioning related to sleep rebound.

BACKGROUND AND AIM: We have previously shown in a rat model of focal cerebral ischemia that sleep deprivation after stroke onset aggravates brain damage. Others reported that sleep deprivation prior to stroke is neuroprotective. The main aim of this study was to test the hypothesis that the neuroprotection may be related to an increase in sleep (sleep rebound) during the acute phase of stroke. METHODS: Male Sprague Dawley rats (n=36) were subjected to continuous polygraphic recordings for baseline, total sleep deprivation (TSD), and 24h after ischemia. TSD for 6h was performed by gentle handling and immediately followed by ischemia. Focal cerebral ischemia was induced by permanent occlusion of distal branches of the middle cerebral artery. Control experiments included ischemia without SD (nSD) and sham surgery with TSD (n=6/group). RESULTS: Shortly after stroke, the amount of slow wave sleep (SWS) and paradoxical sleep (PS) increased significantly (p<0.05) in the TSD/ischemia, resulting in an increase in the total sleep time by 30% compared to baseline, or by 20% compared with the nSD/ischemia group. The infarct volume decreased significantly by 50% in the TSD/ischemia compared to nSD group (p<0.02). Removal of sleep rebound by allowing TSD-rats sleep for 24h before ischemia eliminated the reduction in the infarct size. CONCLUSION PRESTROKE: Sleep deprivation results in sleep rebound and reduces brain damage. Sleep rebound may be causally related to the neuroprotection.

Stimulation of the human RAD51 nucleofilament restricts HIV-1 integration in vitro and in infected cells.

Stable HIV-1 replication requires the DNA repair of the integration locus catalyzed by cellular factors. The human RAD51 (hRAD51) protein plays a major role in homologous recombination (HR) DNA repair and was previously shown to interact with HIV-1 integrase (IN) and inhibit its activity. Here we determined the molecular mechanism of inhibition of IN. Our standard in vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving an IN-DNA complex dissociation mechanism. Furthermore we show that this inhibition mechanism can be promoted in HIV-1-infected cells by chemical stimulation of the endogenous hRAD51 protein. This hRAD51 stimulation induced both an enhancement of the endogenous DNA repair process and the inhibition of the integration step. Elucidation of this molecular mechanism leading to the restriction of viral proliferation paves the way to a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity and highlights the functional interaction between HIV-1 IN and hRAD51.

Selection for increased body length in Subantarctic fur seals on Amsterdam Island.

The traditional distinction between ecological and evolutionary times is eroding, calling for tighter links between ecology and evolution. An example of such a brigde between the two disciplines is the so-called 'animal model', a methodology initially developed by animal breeders, which has become very popular among ecologists studying contemporary microevolution. Using a Bayesian multi-trait 'animal model', we investigated the quantitative genetics of body size, a fitness-related trait, in Subantarctic fur seals (Arctocephalus tropicalis) breeding on Amsterdam Island, Southern Ocean. Our approach jointly modelled the growth and selection processes at work in this population. Body length is heritable for both sexes, and females are under selection for increased body length in this population. We strongly suspect the peculiar ecological context of impoverished, suitable prey availability exacerbated by density-dependence phenomena to be an important selective agent on females breeding on Amsterdam Island.

mRNA-based cancer vaccine: prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes.

Immunization with mRNA encoding tumor antigen is an emerging vaccine strategy for cancer. In this paper, we demonstrate that mice receiving systemic injections of MART1 mRNA histidylated lipopolyplexes were specifically and significantly protected against B16F10 melanoma tumor progression. The originality of this work concerns the use of a new tumor antigen mRNA formulation as vaccine, which allows an efficient protection against the growth of a highly aggressive tumor model after its delivery by intravenous route. Synthetic melanoma-associated antigen MART1 mRNA was formulated with a polyethylene glycol (PEG)ylated derivative of histidylated polylysine and L-histidine-(N,N-di-n-hexadecylamine)ethylamide liposomes (termed histidylated lipopolyplexes). Lipopolyplexes comprised mRNA/polymer complexes encapsulated by liposomes. The tumor protective effect was induced with MART1 mRNA carrying a poly(A) tail length of 100 adenosines at an optimal dose of 12.5 microg per mouse. MART1 mRNA lipopolyplexes elicited a cellular immune response characterized by the production of interferon-gamma and the induction of cytotoxic T lymphocytes. Finally, the anti-B16 response was enhanced using a formulation containing both MART1 mRNA and MART1-LAMP1 mRNA encoding the antigen targeted to the major histocompatibility complex class II compartments by the lysosomal sorting signal of LAMP1 protein. Our results provide a basis for the development of mRNA histidylated lipopolyplexes for cancer vaccine.

Physical interaction of apoptosis-inducing factor with DNA and RNA.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which upon apoptosis induction translocates to the nucleus where it interacts with DNA by virtue of positive charges clustered on the AIF surface. Here we show that the AIF interactome, as determined by mass spectroscopy, contains a large panel of ribonucleoproteins, which apparently bind to AIF through the RNA moiety. However, AIF is devoid of any detectable RNAse activity both in vitro and in vivo. Recombinant AIF can directly bind to DNA as well as to RNA. This binding can be visualized by electron microscopy, revealing that AIF can condense DNA, showing a preferential binding to single-stranded over double-stranded DNA. AIF also binds and aggregates single-stranded and structured RNA in vitro. Single-stranded poly A, poly G and poly C, as well double-stranded A/T and G/C RNA competed with DNA for AIF binding with a similar efficiency, thus corroborating a computer-calculated molecular model in which the binding site within AIF is the same for distinct nucleic acid species, without a clear sequence specificity. Among the preferred electron donors and acceptors of AIF, nicotine adenine dinucleotide phosphate (NADP) was particularly efficient in enhancing the generation of higher-order AIF/DNA and AIF/RNA complexes. Altogether, these data support a model in which a direct interaction of AIF contributes to the compaction of nucleic acids within apoptotic cells.

Investigation of radiation damage in DNA by using atomic force microscopy.

The effect of radiations on supercoiled plasmid DNA has been investigated by using atomic force microscopy (AFM). The DNA molecules were deposited on a substrate and observed by AFM. Alternatively, DNA at different scavenger concentrations was initially exposed to different types of radiations (alpha and X rays) at various doses. After irradiation, fragments (open circular and linearised strands) were observed corresponding to single strand breaks and double strand breaks in DNA. This result indicates the capabilities of AFM for the qualitative detection of strand modifications due to irradiation. The amount of each class of topology enables a quantitative response to be determined for both types of radiation (alpha, X). A value of the radiosensitivity of DNA was obtained as a function of the scavenger concentration. Strong accordance was found between AFM results and those obtained by use of gel electrophoresis. The advantage of AFM in comparison with traditional techniques is the possibility of analysing the radiation effects on one molecule. Indeed, taking the example of alpha particles, it is shown that it is easy to measure the sizes of linear strands by AFM. Such additional or even precise results are difficult to obtain with gel electrophoresis since, in such a case, data are lost through smearing.

Human immunodeficiency virus type 1 central DNA flap: dynamic terminal product of plus-strand displacement dna synthesis catalyzed by reverse transcriptase assisted by nucleocapsid protein.

To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the proviral DNA generating a 99-nucleotide DNA flap (6). This step, catalyzed by reverse transcriptase (RT), is defined as a discrete strand displacement (SD) synthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Using recombinant HIV-1 RT and a circular single-stranded DNA template harboring the cPPT-CTS sequence, we have developed an SD synthesis-directed in vitro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), the main accessory protein of the reverse transcription complex. A full-length HIV-1 central DNA flap was then carried out in vitro. Its synthesis appears faster in the presence of the HIV-1 NCp or the T4-encoded SSB protein (gp32). Finally, a high frequency of strand transfer was shown during the SD synthesis along the cPPT-CTS site with RT alone. This reveals a local and efficient 3'-5' branch migration which emphasizes some important structural fluctuations within the flap. These fluctuations may be stabilized by the NCp chaperone activity. The biological implications of the RT-directed NCp-assisted flap synthesis are discussed within the context of reverse transcription complexes, assembly of the preintegration complexes, and nuclear import of the HIV-1 proviral DNA to the nucleus toward their chromatin targets.

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R.

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.

Static curvature and flexibility measurements of DNA with microscopy. A simple renormalization method, its assessment by experiment and simulation.

We present the derivation of equations based on statistical polymer chain analysis and a method to quantify the average angle value of intrinsic bends and the local flexibility at a given locus on DNA fragments imaged by electron microscopy. DNA fragments of n base-pairs are considered as stiff chains of n jointed unit rigid rods. If the DNA fragments are composed of two branches A0Am and A0Bn, with, respectively, m and n base-pairs, where the standard deviations of the angle formed by two consecutive base-pairs are uniform over each branch, respectively, sigmathetaA and sigmathetaB, we show that the standard deviation of the angle AmA0Bn is: [formula: see text] where sigmatheta0 is the standard deviation of the angle at locus A0. This equation is established for small angular deviations by analysis of DNA at different scales and the validity of the methodology is controlled with the computation of the reduced chi2 statistical test. The length of the DNA fragments must be of the order of, or below, the persistence length, as determined by sets of statistics from computer simulations of DNA fragments. This is verified experimentally by a detailed analysis of the digitized contours of homogeneous linear 139 base-pair DNA fragments observed by electron microscopy. The images are compared to the reconstruction of DNA fragments from the measurements. The value found, sigma0=4.6 degrees/bp, is consistent with the well-accepted value for DNA in a plane. We discuss the relationship between the standard deviation of the measured angles and the flexibility at the base-pair level. This method is useful to quantify directly from microscopy techniques, such as electron or scanning force microscopy, the true bending angle, either intrinsic or induced by a ligand, and its associated flexibility at a given locus in any small DNA fragment.

DNA bending induced by the archaebacterial histone-like protein MC1.

The conformational changes induced by the binding of the histone-like protein MC1 to DNA duplexes have been analyzed by dark-field electron microscopy and polyacrylamide gel electrophoresis. Visualisation of the DNA molecules by electron microscopy reveals that the binding of MC1 induces sharp kinks. Linear DNA duplexes (176 bp) which contained a preferential site located at the center were used for quantitative analysis. Measurements of the angle at the center of all duplexes, at a fixed DNA concentration, as a function of the MC1 concentration, were very well fitted by a simple model of an isotropic flexible junction and an equilibrium between the two conformations of DNA with bound or unbound MC1. This model amounts to double-folded Gaussian distributions and yields an equilibrium deflection angle of theta0=116 degrees for the DNA with bound MC1. It allowed measurements of the fraction of DNA with bound MC1 to be taken as a function of MC1 concentrations and yields an equilibrium dissociation constant of Kd=100 nM. It shows that the flexibility of DNA is reduced by the binding of MC1 and the formation of a kink. The equilibrium dissociation constant value was corroborated by gel electrophoresis. Control of the model by the computation of the reduced chi2 shows that the measurements are consistent and that electron microscopy can be used to quantify precisely the DNA deformations induced by the binding of a protein to a preferential site.

Properties and growth mechanism of the ordered aggregation of a model RNA by the HIV-1 nucleocapsid protein: an electron microscopy investigation.

NCp7, the nucleocapsid protein of the human immunodeficiency virus type 1, induces an ordered aggregation of RNAs, a mechanism that is thought to be involved in the NCp7-induced promotion of nucleic acid annealing. To further investigate this aggregation the morphology and the properties of the NCp7-induced aggregates of the model RNA homoribopolymer, polyA, were investigated by electron microscopy in various conditions. In almost all the tested conditions, the aggregates were spherical and consisted of a central dense core surrounded by a less dense halo made of NCp7-covered polyA molecules. The formation of these aggregates with a narrow distribution of sizes constitutes a distinctive feature of NCp7 over other single-stranded nucleic acid binding proteins. In most conditions, at the shortest times that can be reached experimentally, all the polyA molecules were already incorporated in small aggregates, suggesting that the nucleation step and the first aggregation events took place rapidly. The aggregates then orderly grew with time by fusion of the smaller aggregates to give larger ones. The aggregate halo was important in the fusion process by initiating the bridging between the colliding aggregates. In the presence of an excess of protein, the aggregates grew rapidly but were loosely packed and dissociated easily, suggesting adverse protein-protein interactions in the aggregates obtained in these conditions. In the presence of an excess of nucleotides, the presence of both amorphous nonspherical and slowly growing spherical aggregates suggested some changes in the mechanism of aggregate growth due to an incomplete covering of polyA molecules by NCp7. Finally, we showed that in the absence of added salt, the aggregate fusions were unfavored but not the initial events giving the first aggregates, the reverse being true in the presence of high salt concentrations (> or = 300 mM).

[Complementarity of microscopies in the structural analysis of DNA minicircles associated to protein MC1]. / Complémentarité des microscopies dans l'analyse structurale de minicercles d'ADN associés à la protéine MC1.

Electron microscopy of DNA, either free or complexed with ligands, allows the analysis of local conformational variations along individual molecules. Electron microscopy is unique, in that it has the capacity to determine the average behaviour of a population of molecules observed individually, and can thus provide a better appreciation of variability within the series of molecules than biophysical or biochemical methods. Very encouraging results have been obtained by cryoelectron and near-field microscopies, especially atomic force microscopy, in parallel with traditional techniques for visualizing DNA molecules adsorbed onto a support film. Differences in sample processing procedures and image formation modes render these 3 types of microscopies complementary. The torsional stress of a DNA molecule together with a local curvature induced by the protein MC1 from archaebacteria, can be detected within minicircles comprising 207 base pairs.

Conformational changes of DNA minicircles upon the binding of the archaebacterial histone-like protein MC1.

Binding of the archaebacterial histone-like protein MC1 to DNA minicircles has been examined by gel retardation and electron microscopy. MC1 preferentially binds to a 207-base pair relaxed DNA minicircle as compared with the linear fragment. Random binding is observed at very low ionic strength, and a slight increase in salt concentration highly favors the formation of a complex that corresponds to the binding of two MC1 molecules per DNA ring. Measurements of dissociation rates show that this complex is remarkably stable, and electron microscopy reveals that it is characterized by two diametrically opposed kinks. These results are discussed in regard to the mechanisms by which MC1 affects DNA structure.

Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.

The Fur (ferric uptake regulation) protein is a global regulator that, in the presence of Fe2+, represses the expression of a number of iron-acquisition genes and virulence determinants such as toxins. Dark-field electron microscopy of positively stained Fur-DNA complexes in addition to atomic force microscopy allowed direct visualization of Fur interactions with the regulatory regions of aerobactin and hemolysin operons and provided complementary information about the structure of the complexes. According to the DNA used and the protein/DNA ratio, Fur binding to DNA results in partial or total covering of the fragments, indicating that the protein initiates polymerization along the DNA molecules at specific sites. Negative staining of Fur-DNA complexes revealed a well-ordered structure of the polymer suggesting a helical arrangement. Local rigidification of the DNA molecules resulting from Fur binding could be involved in the repression process.

Fur (ferric uptake regulation) protein interaction with target DNA: comparison of gel retardation, footprinting and electron microscopy analyses.

Fur-DNA interactions were analyzed within the regulatory regions of aerobactin and hemolysin operons by a combination of biochemical and ultrastructural methods. Cartography of the Fur binding sites, carried out from electron micrographs, agreed with the data obtained by DNase I footprinting. Visualization of the complexes confirmed the specificity and metal-dependence of Fur binding and demonstrated that the protein polymerizes on its binding sites. Such a polymerization could be involved in the repression process of the bacterial regulator.

Conformational analysis of a 139 base-pair DNA fragment containing a single-stranded break and its interaction with human poly(ADP-ribose) polymerase.

The conformational changes induced by the introduction of a central and unique single-stranded break in a 139 base-pair DNA duplex have been analysed by means of polyacrylamide gel electrophoresis, HPLC and dark-field electron microscopy. Compared to the control DNA, the disruption of the covalent sugar-phosphate backbone induces a retardation detected both by gel electrophoresis and anion exchange based HPLC. Electron microscopic visualization of the DNA molecules reveals that most of them present a central fracture at the position of the nick. Measures of the angle at the apex were very well fitted by a simple model of isotropic flexible junction assuming spatial Hooke's law and simple basic Boltzmann statistics. This amounts to using a folded Gaussian distribution. The fit yields an angle equilibrium value phi 0 = 122 degrees for the nicked fragment. The angle distribution could also result from an equilibrium between two forms of the molecule with isotropic flexibility at the nicked site: a stacked and a very flexible unstacked form. The majority of bound poly(ADP-ribose) polymerase, a zinc-finger enzyme involved in DNA break detection, was localized at the apex of the V-shaped DNA duplex, with an accentuation of its general V-shaped conformation (phi 0 = 102 degrees).

Quantitative electron microscopic analysis of DNA-protein interactions.

Electron microscopy offers a unique potentiality to visualize individual molecules. For the last 30 years it has been used to study the structure and the interactions of various biological macromolecules. The contribution of electron microscopy is important because of its capacity to demonstrate the existence of conformational structures such as kinks, bents, loops, etc., either on naked DNA, or on DNA associated with various proteins or ligands. Increasing interest was given to such observations when it was found that they provide a direct visualization of interacting molecules involved in DNA metabolism and gene regulation. Technical advances in the preparation of the specimens, their observation in the electron microscope, and the image processing by computers have allowed the shifting from qualitative to quantitative analysis, as illustrated by a few examples from our laboratory.