Science 265 Research Articles

Aquaporins in peripheral nociception

Aquaporins in peripheral nociception

Journeys into the genome of cancer cells

I come from a family in which there have been no scientists or doctors. I was interested, however, in biology at school and started my scientific career by training in medicine at Oxford University and Guys Hospital, London. Practising as a doctor reinforced my curiosity about the biological processes underlying human disease. As a consequence, I pursued a clinical vocation in histopathology, a discipline that couples exposure to the sights and smells of the autopsy room with a daily journey into the often beautiful, sometimes ugly world of healthy and diseased human tissues under the microscope. After an introduction to general histopathology in Nick Wright's department at the Hammersmith Hospital, London, I completed my postgraduate medical training in neuropathology with Peter Lantos at the Maudsley Hospital, London.Peering at the nuclei of cancer cells under the microscope, for me it was a matter of fascination that hidden within them were the key events converting normal cells into cancer cells, and frustration because they were out of reach. Many of the tissue samples examined by pathologists are from cancers. The clonal theory of cancer development and the general role of DNA mutations in generating cancer cell clones had been established by 1986 when I was working as a junior pathologist. Indeed, the first mutated cancer gene, HRAS , had recently been identified through application of the, then new, technologies of recombinant DNA technology. Peering at the nuclei of cancer cells under the microscope, for me it was a matter of fascination that hidden within them were the key events converting normal cells into cancer cells, and frustration because they were out of reach. So, I took 3 years break from medicine to study for a PhD, learning the methods and thinking of molecular oncology in Colin Cooper's laboratory at the Institute …

Abstract 3275: miR-194 counterbalances transcriptional activation of the anti-angiogenic factor thrombospondin-1 by p53

Abstract Thrombospondin-1 (TSP-1) is a key endogenous inhibitor of angiogenesis, negatively regulated by oncogenes (e.g., c-Myc) and transcriptionally activated by the key tumor suppressor p53 (1). Yet in some cancers, such as colon adenocarcinomas, tumor progression accompanied by the loss of p53 does not result in TSP-1 down-regulation (2, 3). This paradox could involve promoter-independent mechanisms, for example microRNA-mediated regulation. In order to test this hypothesis, we conducted a global screen for microRNAs that regulate TSP-1 levels. Gain-of-function experiments and microarray data showed that in addition to the previously identified mir-17-92 cluster members (4, 5), several other miRs, including let-7, mir-199a-3p, mir-218 and miR-194, downregulate TSP-1. mir-194 was of particular interest, since its expression is known to be largely colon-specific (6), maintained by p53 and thus frequently reduced in advanced cancers (7). This reduced miR-194 expression could account for unexpectedly high TSP-1 levels in p53-null tumors. Indeed, mir-194 inhibition with antisense 2’-O-methyl ribonucleotides in p53-sufficient colon cancer cell lines strongly restored TSP-1 levels. Conversely, miR-194 mimics reduced TSP-1 expression at both RNA and protein levels. Furthermore, dual luciferase sensor assay demonstrated that this regulation was mediated by the single mir-194 site in the 3’UTR of the thbs1 gene. We then set up a retroviral transduction-based system to test the involvement of this miR-194 binding site in TSP-1 regulation by p53. Viruses expressing the thrombospondin-1 gene with 3’UTR in either wild type or miR-194-mutated configurations were introduced into HCT116-p53 sufficient cells (miR-194 levels are high) and their p53-null derivatives (miR-194 levels are low). In parental HCT116 cells, the mutation in the mir-194 site sharply increased TSP-1 levels, resulting in reduced angiogenesis in Matrigel plugs and tumor xenografts. However, the effects of this mutation disappeared in the absence of p53, resulting in lower TSP-1 expression levels. These results are consistent with the idea that miR-194 counterbalances transcriptional activation of TSP-1 by p53 and underscore the complex relationship between promoter-dependent and microRNA-mediated effects of p53. Grant Support: R01 CA122334 (ATT) and T32 CA009140 (PS) 1. K. M. Dameron, O. V. Volpert, M. A. Tainsky, N. Bouck, Science 265, 1582 (1994). 2. S. Kaiser et al., Genome Biol 8, R131 (2007). 3. D. A. Notterman, U. Alon, A. J. Sierk, A. J. Levine, Cancer Res 61, 3124 (2001). 4. M. Dews et al., Nat Genet 38, 1060 (2006). 5. M. Dews et al., Cancer Res 70, 8233 (2010). 6. K. Hino et al., RNA. 14, 1433 (2008). 7. C. J. Braun et al., Cancer Res 68, 10094 (2008). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3275. doi:10.1158/1538-7445.AM2011-3275

Resolution of Respect: Professor Paul Schultz Martin 1928–2010

Resolution of Respect: Professor Paul Schultz Martin 1928–2010

Breakup of diminutive Rayleigh jets

Discharging a liquid from a nozzle at sufficient large velocity leads to a continuous jet that due to capillary forces breaks up into droplets. Here we investigate the formation of microdroplets from the breakup of micron-sized jets with ultra high-speed imaging. The diminutive size of the jet implies a fast breakup time scale τc=ρr3/γ of the order of 100 ns, and requires imaging at 14×106 frames/s. We directly compare these experiments with a numerical lubrication approximation model that incorporates inertia, surface tension, and viscosity [J. Eggers and T. F. Dupont, J. Fluid Mech. 262, 205 (1994); X. D. Shi, M. P. Brenner, and S. R. Nagel, Science 265, 219 (1994)]. The lubrication model allows to efficiently explore the parameter space to investigate the effect of jet velocity and liquid viscosity on the formation of satellite droplets. In the phase diagram, we identify regions where the formation of satellite droplets is suppressed. We compare the shape of the droplet at pinch-off between the lubrication approximation model and a boundary-integral calculation, showing deviations at the final moment of the pinch-off. In spite of this discrepancy, the results on pinch-off times and droplet and satellite droplet velocity obtained from the lubrication approximation agree with the high-speed imaging results.

A structural model for the single-stranded DNA genome of filamentous bacteriophage Pf1.

The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament ( approximately 2000 x 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue alpha-helical subunit. The subunit alpha-helices, which are inclined at a small average angle ( approximately 16 degrees ) from the virion axis, are arranged compactly around the DNA core. Orientations of the Pf1 DNA nucleotides with respect to the filament axis are not known. In this work we report and interpret the polarized Raman spectra of oriented Pf1 filaments. We demonstrate that the polarizations of DNA Raman band intensities establish that the nucleotide bases of packaged Pf1 DNA are well ordered within the virion and that the base planes are positioned close to parallel to the filament axis. The present results are combined with a previously proposed projection of the intraviral path of Pf1 DNA [Liu, D. J., and Day, L. A. (1994) Science 265, 671-674] to develop a novel molecular model for the Pf1 assembly.

Structure and dynamics of bioactive phosphosilicate glasses and melts fromab initiomolecular dynamics simulations

Ab initio (Car-Parrinello) molecular dynamics simulations were carried out to investigate the melt precursor of a modified phosphosilicate glass with bioactive properties, and to quench the melt to the vitreous state. The properties of the 3000 K liquid were extensively compared with those of the final glass structure. The melt is characterized by a significant fraction of structural defects (small rings, undercoordinated and overcoordinated ions), often combined together. The creation or removal of these coordinative defects in the liquid (through Si-O bond formation or dissociation) reflects frequent exchanges within the silicate first coordination shell, which in turn dynamically modify the intertetrahedral connectivity of silicate groups. The observed dynamical variation in both the identity and the number of silicate groups linked to a tagged Si (Q(n) speciation) are considered key processes in the viscous flow of silicate melts [I. Farnan and J. F. Stebbins, Science 265, 1206 (1994)]. On the other hand, phosphate groups do not show an equally marked exchange activity in the coordination shell, but can still form links with Si. Once formed, these Si-O-P bridges are rather stable, and in fact they are retained in the glass phase obtained after cooling; their formation within the present full ab initio melt-and-quench approach strongly supports their presence in melt-derived phosphosilicate glasses with bioactive applications. On the other hand, the simulations show that the fraction of structural defects rapidly decreases during the cooling, and the glass is essentially free of miscoordinated ions and small rings.

Computational aspects of Worm-Like-Chain interpolation formulas

We show that the polynomial Worm-Like-Chain (WLC) interpolation formula introduced in [C. Bouchiat, M.D. Wang, J.-F. Allemand, T. Strick, S.M. Block, V. Croquette, Estimating the persistence length of a Worm-Like Chain molecule from force–extension measurements, Biophys. J. 76 (1) (1999) 409–413] in order to improve data fitting with respect to the WLC interpolation formula in [C. Bustamante, J.F. Marko, E.D. Siggia, S. Smith, Entropic elasticity of lambda-phage DNA, Science 265 (1994) 1599. Technical comment] is not unique. Ill-conditioning of the over-determined linear system associated with interpolation of synthetic data from [C. Bouchiat, M.D. Wang, J.-F. Allemand, T. Strick, S.M. Block, V. Croquette, Estimating the persistence length of a Worm-Like Chain molecule from force–extension measurements, Biophys. J. 76 (1) (1999) 409–413] is highlighted. Moreover, if the coefficients in the associated polynomial correction are considered as free parameters in the least squares fitting procedure for actual experimental data, then more than one solution with a low residual can be identified. For these reasons we propose modification of the interpolation formula in [C. Bustamante, J.F. Marko, E.D. Siggia, S. Smith, Entropic elasticity of lambda-phage DNA, Science 265 (1994) 1599. Technical comment] so that, for moderate extensions, quadratic contributions are included and no additional parameters are required. We show that relative errors for the fit of synthetically generated data are less than 2% and for actual data they are comparable with and sometimes better than those obtained by using polynomial WLC interpolation formulas.

On the stretching vibrations of linear C 13: A coupled cluster study

The quadratic stretching force constants and the harmonic stretching vibrations of linear C 13 have been studied by the coupled cluster variant CCSD(T) in conjunction with large basis sets. A large absolute IR intensity of 22,218 km mol −1 is calculated for the ν 9 band. In comparison with a high-resolution spectroscopic study [T.F. Giesen, A. van Orden, H.J. Hwang, R.S. Fellers, R.A. Provencal, R.J. Saykally, Science 265 (1994) 756], the corresponding wavenumber experiences an unusually large anharmonicity contribution of 7%.

A Bex‐cycle built for two

Neurotrophins promote neuronal survival, but it has been recognized that they also stimulate differentiation. Indeed, in early studies by Hamburger, Levi‐Montelcini and Cohen, nerve growth factor (NGF) not only promoted survival, but also caused marked neurite outgrowth when added to explants of sensory ganglia (Cohen et al , 1954). The ability of NGF to promote differentiation was particularly well illustrated when Greene & Tischler (1976) showed that a pheochromocytoma cell line (PC12) treated with NGF exited the cell cycle and differentiated into a sympathetic neuron‐like phenotype. Since then, it has been established that the survival and differentiation effects of the neurotrophins are primarily mediated by binding to members of the Trk family of tyrosine kinase receptors (Huang & Reichardt, 2003); however, the first receptor identified for NGF was a 75‐kDa protein referred to as p75 (Chao et al , 1986; Radeke et al , 1987). In the past few years, the p75 receptor has received a lot of attention because of its multifunctional signalling ability. This receptor can promote cell survival or induce apoptosis, regulate neurite outgrowth and promote peripheral myelination, depending on the specific cellular context, the expression of co‐receptors and the activating ligand (Barker, 2004). The role of p75 in promoting differentiation and cell‐cycle arrest has been largely overlooked in recent years, even though this was one of the first cellular responses to be shown to result from the binding of NGF to the receptor. Long considered to be simply a co‐receptor for the Trks, p75 came to be recognized as a bona fide signalling molecule when it was shown to activate sphingomyelinase. This resulted in the production of ceramide, which led to the differentiation of T9 glioma cells as indicated by their cell‐cycle arrest and process outgrowth (Dobrowsky et al , 1994). Although the focus of most subsequent research …

Short exposure to an enriched environment accelerates plasticity in the barrel cortex of adult rats

Cortical sensory neurons adapt their response properties to use and disuse of peripheral receptors in their receptive field. Changes in synaptic strength can be generated in cortex by simply altering the balance of input activity, so that a persistent bias in activity levels modifies cortical receptive field properties. Such activity-dependent plasticity in cortical cell responses occurs in rat cortex when all but two whiskers are trimmed for a period of time at any age. The up-regulation of evoked responses to the intact whiskers is first seen within 24 h in the supragranular layers [Diamond ME, Huang W, Ebner FF (1994) Laminar comparison of somatosensory cortical plasticity. Science 265(5180):1885–1888] and continues until a new stable state is achieved [Diamond ME, Armstrong-James M, Ebner FF (1993) Experience-dependent plasticity in adult rat barrel cortex. Proc Natl Acad Sci U S A 90(5):2082–2086; Armstrong-James M, Diamond ME, Ebner FF (1994) An innocuous bias in whisker use in adult rat modifies receptive fields of barrel cortex neurons. J Neurosci 14:6978–6991]. These and many other results suggest that activity-dependent changes in cortical cell responses have an accumulation threshold that can be achieved more quickly by increasing the spike rate arising from the active region of the receptive field. Here we test the hypothesis that the rate of neuronal response change can be accelerated by placing the animals in a high activity environment after whisker trimming. Test stimuli reveal an highly significant receptive field bias in response to intact and trimmed whiskers in layer IV as well as in layers II–III neurons in only 15 h after whisker trimming. Layer IV barrel cells fail to show plasticity after 15–24 h in a standard cage environment, but produce a response bias when activity is elevated by the enriched environment. We conclude that elevated activity achieves the threshold for response modification more quickly, and this, in turn, accelerates the rate of receptive field plasticity.

Late Pleistocene and Holocene paleoseismology of an intraplate seismic zone in a large alluvial valley, the New Madrid seismic zone, Central USA

Late Pleistocene and Holocene paleoseismology of an intraplate seismic zone in a large alluvial valley, the New Madrid seismic zone, Central USA

Jupiter's atmospheric temperatures: From Voyager IRIS to Cassini CIRS

Retrievals performed on Cassini Composite Infrared Spectrometer data obtained during the distant Jupiter flyby in 2000/2001 have been used to generate global temperature maps of the planet in the troposphere and stratosphere, but to higher latitudes than were shown previously by Flasar et al. [Flasar, F.M., 39 colleagues, 2004a. Nature 427, 132–135; Flasar, F.M., 44 colleagues, 2004b. Space Sci. Rev. 115, 169–297]. Similar retrievals were performed on Voyager 1 IRIS data to provide the first detailed IRIS map of the stratosphere, and high latitudes in the troposphere. Thermal winds were calculated for each data set and show strong vertical shears in the zonal winds at low latitudes, and meridional temperature gradients indicate the presence of circumpolar jets, as well. The temperatures retrieved from the two spacecraft were also compared with yearly ground-based data obtained over the intervening two decades. Tropospheric temperatures reveal gradual changes at low latitudes, with little obvious seasonal or short-term variation [Orton et al., 1994. Science 265, 625–631]. Stratospheric temperatures show much more complicated behavior over short timescales, consistent with quasi-quadrennial oscillations at low latitudes, as suggested in prior analyses of shorter intervals of ground-based data [Orton et al., 1991. Science 252, 537–542; Friedson, A.J., 1999. Icarus 137, 34–55]. A scaling analysis indicates that meridional motions, mechanically forced by wave or eddy convergence, play an important role in modulating the temperatures and winds in the upper troposphere and stratosphere on seasonal and shorter timescales. At latitudes away from the equator, the mechanical forcing can be derived simply from a temporal record of temperature and its vertical derivative. Ground-based observations with improved vertical resolution and/or long-term monitoring from spacecraft are required for this purpose, though the Voyager and Cassini data given indications that the magnitude of the forcing is very small.

Water-load definition in the glacio-hydro-isostatic sea-level equation

Models of glacio-hydro-isostatic rebound and the concomitant sea-level change have been progressively improved over the past three decades. Recently, the procedures used by the group at the Australian National University (ANU) for the hydro-isostatic component of the theory have been questioned (Quat. Sci. Rev. 21 (2002) 409) although the details of the ANU group's procedures have not been published because they are mainly computational in nature rather than representing significant conceptual advances. Because of this criticism, we set out here in detail the procedures that have been used for the treatment of the migration of shorelines as sea levels rise and fall, the effect of retreat and advancing grounded ice on shelves and shallow seas, and the transitions from grounded to floating ice (and vice versa). We conclude that there is no basis for the criticism, that these formulations and their implementation provide a high resolution and complete description of both sea-level change and of the estimates of volumes of ice exchanged with the oceans. The results from this formulation are confirmed by the entirely independent analyses of Milne et al. (Quat. Sci. Rev. 21 (2002) 361) and Mitrovica and Milne (Geophys. J. Int. (2002), submitted for publication) who conclude that our formulation is significantly more accurate than the procedure advocated by Peltier (Science 265 (1994) 195; Rev. Geophys. 36 (1998a) 603, Geophys. Res. Lett. 25 (1998b) 3955).

Assessment of induction of biliverdin reductase in a mouse model of middle cerebral artery occlusion

Reproducible animal models of stroke are indispensable for investigation of pathogenesis and treatment of ischemic brain injury. Defined location and size of infarction as well as consistent production of neurological deficits make it possible to evaluate therapeutic potential of neuroprotective agents as well as to assess the impact of gene deletion [Nat. Med. 3 (1997) 1089; Science 265 (1994) 1883; Nat. Med. 4 (1998) 228] or overexpression [J. Neurochem. 72 (1999) 1187; J. Neurosci. 17 (1997) 7655] on neuroprotection in genetically altered mice. Ischemic stroke in mice can be reliably replicated by means of an open craniectomy exposure followed by permanent occlusion of the trunk and branches of the middle cerebral artery (MCA). Open craniectomy model is known to be statistically robust, yielding a coefficient of variation of <10%, and requiring minimal number of animals to validate the concept of statistical power. In the past, this model as well as some of its variants had been used in pivotal scientific studies to demonstrate impact of therapeutic genes on the course of ischemic neuronal injury [Neuron 13 (1994) 1017], as well as identification of ‘culprit genes’ responsible for progression of ischemic injury [J. Cerebr. Blood Flow Metab. 14 (1994) 887; Science 265 (1994) 1883; J. Neurosci. 17 (1997) 7655] through continuous recruitment of marginally ischemic penumbra into ischemic core [Trends Neurosci. 22 (1999) 391]. This protocol describes mapping of the ischemic penumbra using NADPH diaphorase staining as well as assessment of penumbral endogenous antioxidant reserves by detection of cellular biliverdin reductase mRNA and protein levels using immunocytochemistry and in situ hybridization histological techniques.

Selective expression of Cre recombinase in skeletal muscle fibers

genesisVolume 26, Issue 2 p. 165-166 ArticleFree Access Selective expression of Cre recombinase in skeletal muscle fibers Gerald W. M. Bothe, Corresponding Author Gerald W. M. Bothe Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkTaconic Biotechnology, One University Place, Rensselaer, New York 12144Search for more papers by this authorJeffrey A. Haspel, Jeffrey A. Haspel Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this authorCynthia L. Smith, Cynthia L. Smith Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this authorHeidi H. Wiener, Heidi H. Wiener Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this authorSteven J. Burden, Steven J. Burden Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this author Gerald W. M. Bothe, Corresponding Author Gerald W. M. Bothe Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkTaconic Biotechnology, One University Place, Rensselaer, New York 12144Search for more papers by this authorJeffrey A. Haspel, Jeffrey A. Haspel Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this authorCynthia L. Smith, Cynthia L. Smith Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this authorHeidi H. Wiener, Heidi H. Wiener Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this authorSteven J. Burden, Steven J. Burden Skirball Institute for Molecular Medicine, New York University Medical Center, New York, New YorkSearch for more papers by this author First published: 23 February 2000 https://doi.org/10.1002/(SICI)1526-968X(200002)26:2<165::AID-GENE22>3.0.CO;2-FCitations: 109AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. LITERATURE CITED Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, Accili D, Goodyear LJ, Kahn CR. 1998. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell 2: 559– 569. Burden SJ. 1998. The formation of neuromuscular synapses. Genes Dev 12: 133– 148. Gassmann M, Casagranda F, Orioli D, Simon H, Lai C, Klein R, Lemke G. 1995. Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature 378: 390– 394. Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K. 1994. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265: 103– 106. Lee KF, Simon H, Chen H, Bates B, Hung MC, Hauser C. 1995. Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378: 394– 398. Lyons GE, Ontell M, Cox R, Sassoon D, Buckingham M. 1990. The expression of myosin genes in developing skeletal muscle in the mouse embryo. J Cell Biol 111: 1465– 1476. Meyer D, Birchmeier C. 1995. Multiple essential functions of neuregulin in development. Nature 378: 386– 390. Schmalbruch H, Hellhammer U. 1977. The number of nuclei in adult rat muscles with special reference to satellite cells. Anat Rec 189: 169– 175. Citing Literature Volume26, Issue2Special Issue: Tissue Specific Expression of Cre Recombinase in MiceFebruary 2000Pages 165-166 ReferencesRelatedInformation

Experience-dependent phase-reversal of hippocampal neuron firing during REM sleep

The idea that sleep could serve a cognitive function has remained popular since Freud stated that dreams were “not nonsense” but a time to sort out experiences [S. Freud, Letter to Wilhelm Fliess, May 1897, in The Origins of Psychoanalysis — Personal Letters of Sigmund Freud, M. Bonaparte, A. Freud, E. Kris (Eds.), Translated by E. Mosbacher, J. Strachey, Basic Books and Imago Publishing, 1954]. Rapid eye movement (REM) sleep, which is associated with dream reports, is now known to be is important for acquisition of some tasks [A. Karni, D. Tanne, B.S. Rubenstein, J.J.M. Askenasy, D. Sagi, Dependence on REM sleep of overnight improvement of a perceptual skill, Science 265 (1994) 679–682; C. Smith, Sleep states and learning: a review of the animal literature, Biobehav. Rev. 9 (1985) 157–168]; although why this is so remains obscure. It has been proposed that memories may be consolidated during REM sleep or that forgetting of unnecessary material occurs in this state [F. Crick, G. Mitchison, The function of dream sleep, Nature 304 (1983) 111-114; D. Marr, Simple memory: a theory for archicortex, Philos. Trans. R. Soc. B. 262 (1971) 23–81]. We studied the firing of multiple single neurons in the hippocampus, a structure that is important for episodic memory, during familiar and novel experiences and in subsequent REM sleep. Cells active in familiar places during waking exhibited a reversal of firing phase relative to local theta oscillations in REM sleep. Because firing-phase can influence whether synapses are strengthened or weakened [C. Holscher, R. Anwyl, M.J. Rowan, Stimulation on the positive phase of hippocampal theta rhythm induces long-term potentiation that can be depotentiated by stimulation on the negative phase in area CA1 in vivo, J. Neurosci. 15 (1977) 6470–6477; P.T. Huerta, J.E. Lisman, Bidirectional synaptic plasticity induced by a single burst during cholinergic theta oscillation in CA1 in vitro, Neuron 15 (1995) 1053–1063; C. Pavlides, Y.J. Greenstein, M. Grudman, J. Winson, Long-term potentiation in the dentate gyrus is induced preferentially on the positive phase of theta-rhythm, Brain Res. 439 (1988) 383–387] this experience-dependent phase shift, which developed progressively over multiple sessions in the environment, is consistent with the hypothesis that circuits may be restructured during REM sleep by selectively strengthening recently acquired memories and weakening older ones.

Characterization of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxynitrous acid.

Large RNAs fold into complex structures which determine their biological activities. A full understanding of both RNA structure and dynamics will include the description of the pathways by which these structures are formed. Kinetic footprinting [Sclavi, B., et al. (1997) J. Mol. Biol. 266, 144-159] has been shown to be a powerful method for the study of dynamic processes involving RNA. Here we describe the use of a readily available reagent, peroxynitrous acid, as a kinetic footprinting tool for the study of RNA folding. Hydroxyl radicals generated from this reagent were used to footprint the Tetrahymena ribozyme during its magnesium-dependent folding-in agreement with synchroton X-ray footprinting [Sclavi, B., et al. (1998) Science 279, 1940-1943] and oligonucleotide/hybridization cleavage experiments [Zarrinkar, P. P., and Williamson, J. R. (1994) Science 265, 918-924], this work suggests an ordered, hierarchical folding pathway for the ribozyme. Several slow steps in the folding pathway were observed in the peroxynitrous acid footprinting, but none of these corresponded to the rate-determining step of folding. This suggests that the formation of the global, protected structure is followed by one or more slow local rearrangements to yield the final active structure. These studies illustrate the utility of peroxynitrous acid as a reagent for the elucidation of RNA folding pathways and the study of RNA dynamics.

The core histone N-terminal domains are required for multiple rounds of catalytic chromatin remodeling by the SWI/SNF and RSC complexes.

SWI/SNF and RSC are large, distinct multi-subunit complexes that use the energy of ATP hydrolysis to disrupt nucleosome structure, facilitating the binding of transcription factors or restriction enzymes to nucleosomes [Cote, J., Quinn, J., Workman, J. L., and Peterson, C. L. (1994) Science 265, 53-60 (1); Lorch, Y., Cairns, B. R., Zhang, M., and Kornberg, R. D. (1998) Cell 94, 29-34 (2)]. Here we have used a quantitative assay to measure the activities of these ATP-dependent chromatin remodeling complexes using nucleosomal arrays reconstituted with hypoacetylated, hyperacetylated, or partially trypsinized histones. This assay is based on measuring the kinetics of restriction enzyme digestion of a site located within the central nucleosome of a positioned 11-mer array [Logie, C., and Peterson, C. L. (1997) EMBO J. 16, 6772-6782 (3)]. We find that the DNA-stimulated ATPase activities of SWI/SNF and RSC are not altered by the absence of the histone N-termini. Furthermore, ATP-dependent nucleosome remodeling is also equivalent on all three substrate arrays under reaction conditions where the concentrations of nucleosomal array and either SWI/SNF or RSC are equivalent. However, SWI/SNF and RSC cannot catalytically remodel multiple nucleosomal arrays in the absence of the histone termini, and this catalytic activity of SWI/SNF is decreased by histone hyperacetylation. These results indicate that the histone termini are important for SWI/SNF and RSC function; and, furthermore, our data defines a step in the remodeling cycle where the core histone termini exert their influence. This step appears to be after remodeling, but prior to intermolecular transfer of the remodelers to new arrays.

Stretched and overwound DNA forms a Pauling-like structure with exposed bases.

We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from sigma approximately -0.015 down to sigma = -1. Surprisingly, for positively supercoiled DNA (sigma > +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has approximately 2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling's early DNA structure [Pauling, L. & Corey, R. B. (1953), Proc. Natl. Acad. Sci. USA 39, 84-97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994) Science 265, 671-674].