Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine⋅oligopurine 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 PPT/HIV-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 PPT/HIV-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.
A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.
de Lecea, L.; Kilduff, T. S.; Peyron, C.; Gao, X.-B.; Foye, P. E.; Danielson, P. E.; Fukuhara, C.; Battenberg, E. L. F.; Gautvik, V. T.; Bartlett, F. S.; Frankel, W. N.; van den Pol, A. N.; Bloom, F. E.; Gautvik, K. M.; Sutcliffe, J. G.
Fonte: National Academy of SciencesPublicador: National Academy of Sciences
We describe a hypothalamus-specific mRNA that encodes preprohypocretin, the putative precursor of a pair of peptides that share substantial amino acid identities with the gut hormone secretin. The hypocretin (Hcrt) protein products are restricted to neuronal cell bodies of the dorsal and lateral hypothalamic areas. The fibers of these neurons are widespread throughout the posterior hypothalamus and project to multiple targets in other areas, including brainstem and thalamus. Hcrt immunoreactivity is associated with large granular vesicles at synapses. One of the Hcrt peptides was excitatory when applied to cultured, synaptically coupled hypothalamic neurons, but not hippocampal neurons. These observations suggest that the hypocretins function within the CNS as neurotransmitters.
Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-Fc chimeric proteins show that constructs containing Ig1–3 or Ig1 domains...
Electron microscopy does not, in principle, require highly ordered crystals to determine a high-resolution structure. Nevertheless, crystals of any type help to constrain the molecules into a more limited range of orientations and positions, from which it is easier to carry out structure determination. We describe an improved procedure for determination of crystalline disorder, which we have applied to poorly ordered two-dimensional crystals of the chloride pump halorhodopsin from Halobacterium salinarum. The new image analysis procedure involves the use of a reference projection calculated from a global three-dimensional map to carry out the initial cross-correlation analysis. Coupled with a greater number of images taken with field emission gun microscopes, this has allowed us to calculate a three-dimensional structure for halorhodopsin, in which the seven transmembrane helices and certain molecular features, such as the β-ionone ring of retinal, are now resolved.
Functional activity of N-methyl-d-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although d-serine is a more potent agonist. Localizations of d-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of d-serine with d-amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch–clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied d-serine, which by itself did not potentiate NMDA receptor-mediated synaptic responses. Thus, d-serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses.
Cladistic analysis of cranial and dental evidence has been widely used to generate phylogenetic hypotheses about humans and their fossil relatives. However, the reliability of these hypotheses has never been subjected to external validation. To rectify this, we applied identical methods to equivalent evidence from two groups of extant higher primates for whom reliable molecular phylogenies are available, the hominoids and papionins. We found that the phylogenetic hypotheses based on the craniodental data were incompatible with the molecular phylogenies for the groups. Given the robustness of the molecular phylogenies, these results indicate that little confidence can be placed in phylogenies generated solely from higher primate craniodental evidence. The corollary of this is that existing phylogenetic hypotheses about human evolution are unlikely to be reliable. Accordingly, new approaches are required to address the problem of hominin phylogeny.
Optical single-transporter recording, a recently established fluorescence microscopic method, was used to study the selective transport of proteins through single nuclear pore complexes (NPCs) of Xenopus oocytes. Recombinant proteins containing either a nuclear localization signal (import protein) or a nuclear export signal (export protein) were generated as transport substrates. To approximate in vivo conditions as closely as possible, a Xenopus egg extract was applied to the cytosolic side and a Xenopus oocyte nuclear extract to the nuclear side of the NPCs. It was found that protein transport through functionally isolated, “patched” NPCs depended on signal sequences, extracts, and metabolic energy, as in vivo. All NPCs were competent for both import and export. The transport direction was strictly determined by the transport signal, and at none of the conditions explored was the import protein exported or the export protein imported, even when the application sides of the extracts were reversed. The mean transport rates of the single NPC were ≈2 dimers/s for the import protein and ≈4 dimers/s for the export protein (≈15 μM substrate concentration, 22–24°C), in good agreement with in vivo rates estimated for mammalian cells by microinjection experiments. The study shows that optical single-transporter recording permits the analysis of membrane transport processes not previously accessible to single-transporter recording and thus provides additional possibilities for the elucidation of nucleocytoplasmic transport mechanisms.
Developments of technologies for delivery of foreign genes to the central nervous system are opening the field to promising treatments for human neurodegenerative diseases. Gene delivery vectors need to fulfill several criteria of efficacy and safety before being applied to humans. The ability to drive expression of a therapeutic gene in an adequate number of cells, to maintain long-term expression, and to allow exogenous control over the transgene product are essential requirements for clinical application. We describe the use of an adenovirus vector encoding human tyrosine hydroxylase (TH) 1 under the negative control of the tetracycline-sensitive gene regulatory system for direct injection into the dopamine-depleted striatum of a rat model of Parkinson’s disease. This vector mediated synthesis of TH in numerous striatal cells and transgene expression was observed in a large proportion of them for at least 17 weeks. Furthermore, doxycyline, a tetracycline analog, allowed efficient and reversible control of transgene expression. Thus, the insertion of a tetracycline-sensitive regulatory cassette into a single adenovirus vector provides a promising system for the development of successful and safe therapies for human neurological diseases. Our results also confirm that future effective gene replacement approaches to Parkinson’s disease will have to consider the concomitant transfer of TH and GTP-cyclohydrolase transgenes because the synthesis of the TH cofactor tetrahydrobiopterin may be crucial for restoration of the dopaminergic deficit.
A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H]thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2 and H9 cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover...
Transition path sampling has been applied to the molecular dynamics of the alanine dipeptide in vacuum and in aqueous solution. The analysis shows that more degrees of freedom than the traditional dihedral angles, φ and ψ, are necessary to describe the reaction coordinates for isomerization of this molecule. In vacuum, an additional dihedral angle is identified as significant. In solution, solvent variables are shown to play a significant role, and this role appears to be more specific than can be captured by friction models. Implications for larger molecules are discussed.
Pea weevil (Bruchus pisorum L.) oviposition on pods of specific genetic lines of pea (Pisum sativum L.) stimulates cell division at the sites of egg attachment. As a result, tumor-like growths of undifferentiated cells (neoplasms) develop beneath the egg. These neoplasms impede larval entry into the pod. This unique form of induced resistance is conditioned by the Np allele and mediated by a recently discovered class of natural products that we have identified from both cowpea weevil (Callosobruchus maculatus F.) and pea weevil. These compounds, which we refer to as “bruchins,” are long-chain α,ω-diols, esterified at one or both oxygens with 3-hydroxypropanoic acid. Bruchins are potent plant regulators, with application of as little as 1 fmol (0.5 pg) causing neoplastic growth on pods of all of the pea lines tested. The bruchins are, to our knowledge, the first natural products discovered with the ability to induce neoplasm formation when applied to intact plants.
The capability to enhance or suppress the nucleation of protein crystals opens opportunities in various fundamental and applied areas, including protein crystallography, production of protein crystalline pharmaceuticals, protein separation, and treatment of protein condensation diseases. Herein, we show that the rate of homogeneous nucleation of lysozyme crystals passes through a maximum in the vicinity of the liquid–liquid phase boundary hidden below the liquidus (solubility) line in the phase diagram of the protein solution. We found that glycerol and polyethylene glycol (which do not specifically bind to proteins) shift this phase boundary and significantly suppress or enhance the crystal nucleation rates, although no simple correlation exists between the action of polyethylene glycol on the phase diagram and the nucleation kinetics. The control mechanism does not require changes in the protein concentration, acidity, and ionicity of the solution. The effects of the two additives on the phase diagram strongly depend on their concentration, which provides opportunities for further tuning of nucleation rates.
A broad battery of psychoacoustic measures and standard measures of reading and spelling were applied to 102 adults. The test group included individuals with a childhood history of reading difficulties and controls with no reported reading difficulties. Reading scores were variable in both groups. Poor auditory processing abilities were recorded in poor readers; particular difficulties were posed by tasks requiring spectral distinctions, the simplest of which was pure tone frequency discrimination. In absolute terms, the greatest deficits were recorded in tasks in which stimuli were presented in brief forms and in rapid succession. Auditory processing abilities accounted for more than 50% of the reading score variance in the control group, but their correlation with reading scores was lower in the group with childhood histories of reading difficulties. The additional variability in the latter group resulted largely from the prevalence of reading-compensated poor psychoacoustic performers, whose short-term word memory was also typically poor. Taken together, these findings support a link between impaired auditory resolution and poor reading. Psychoacoustic difficulties are largely retained through adulthood and may be the source of the retained reading difficulties.
The hypothesis that dynamic actin filaments participate in specific aspects of synaptic plasticity was investigated at the Schaffer-collateral-CA1 pyramidal cell synapse of mouse hippocampus. Low concentrations (0.01–1 μM) of compounds that inhibit actin filament assembly were bath applied to hippocampal slices during extracellular recording of field excitatory postsynaptic potentials. Cytochalasin D, cytochalasin B, and latrunculin A all impaired the maintenance of LTP induced by brief high-frequency stimulation. This effect on LTP maintenance was specific, because none of the compounds affected basal synaptic transmission, paired-pulse facilitation, LTP induction, or post-tetanic potentiation. The effect of cytochalasin B was reversible. The results are consistent with a model in which dynamic actin filaments play an essential role in the molecular mechanisms underlying the early maintenance phase of LTP, such as growth of new synaptic connections or conversion of silent synapses.
Medieval Black Death is believed to have killed up to one-third of
the Western European population during the 14th century. It was
identified as plague at this time, but recently the causative organism
was debated because no definitive evidence has been obtained to confirm
the role of Yersinia pestis as the agent of plague. We
obtained the teeth of a child and two adults from a 14th century grave
in France, disrupted them to obtain the pulp, and applied the new
“suicide PCR” protocol in which the primers are used only once.
There were no positive controls: Neither Yersinia nor
Yersinia DNA were introduced in the laboratory. A
negative result is followed by a new test using other primers; a
positive result is followed by sequencing. The second and third primer
pair used, coding for a part of the pla gene, generated
amplicons whose sequence confirmed that it was Y. pestis
in 1 tooth from the child and 19/19 teeth from the adults. Negative
controls were negative. Attempts to detect the putative alternative
etiologic agents Bacillus anthracis and
Rickettsia prowazekii failed. Suicide PCR avoids any
risk of contamination as it uses a single-shot primer—its specificity
is absolute. We believe that we can end the controversy: Medieval Black
Death was plague.
Students of social evolution are concerned not only with the
general course it has followed, but also with the mechanisms that have
brought it about. One such mechanism comes into play when the
quantitative increase in some entity, usually population, reaching a
certain threshold, gives rise to a qualitative change in the structure
of a society. This mechanism, first recognized by Hegel, was seized on
by Marx and Engels. However, neither they nor their current followers
among anthropologists have made much use of it in attempting to explain
social evolution. But as this paper attempts to show, in those few
instances when the mechanism has been invoked, it has heightened our
understanding of the process of social evolution. And, it is argued, if
the mechanism were more widely applied, further understanding of the
course of evolution could be expected to result.
Evolution of protein structure from random coil to native is first
represented topologically by its time-dependent sequences of
discretized Ramachandran basins occupied by successive backbone
residues. Introducing energetic and entropic criteria at each instant
of observation transforms the description from a structurally ambiguous
topological representation to an unambiguous geometric picture of the
folding process. The method is applied with success to folding of
β-lactoglobulin, traditionally perplexing because of its reputed
nonhierarchical folding pattern. This molecule passes through a stage,
ca. 0.1 μs duration, of transient, “flickering”
α-helical structure, until a bit of tertiary structure forms that
stabilizes the system long enough to allow it to pass to its native
Human respiratory syncytial virus (HRSV) is a major cause of a
number of severe respiratory diseases, including bronchiolitis and
pneumonia, in infants and young children. The HRSV F protein, a
glycoprotein essential for viral entry, is a primary target for vaccine
and drug development. Two heptad-repeat regions within the HRSV F
sequence were predicted by the computer program
learncoil-vmf. These regions are thought to form
trimer-of-hairpins–like structures, similar to those found in the
fusion proteins of several enveloped viruses. The hairpin structure
likely brings the viral and cellular membranes into close apposition,
thereby facilitating membrane fusion and subsequent viral entry. Here,
we show that peptides, denoted HR-N and HR-C, corresponding to the
heptad-repeat regions from the N-terminal and C-terminal segments of
the HRSV F protein, respectively, form a stable α-helical trimer of
heterodimers. The HRSV N/C complex was crystallized and its x-ray
structure was determined at 2.3-Å resolution. As anticipated, the
complex is a six-helix bundle in which the HR-N peptides form a
three-stranded, central coiled coil, and the HR-C peptides pack in an
antiparallel manner into hydrophobic grooves on the coiled-coil
surface. There is remarkable structural similarity between the HRSV
N/C complex and the fusion protein core of other viruses...
Electron tomography is the only technique available that allows us
to visualize the three-dimensional structure of unfixed and unstained
cells currently with a resolution of 6–8 nm, but with the prospect to
reach 2–4 nm. This raises the possibility of detecting and identifying
specific macromolecular complexes within their cellular context by
virtue of their structural signature. Templates derived from the
high-resolution structure of the molecule under scrutiny are used to
search the reconstructed volume. Here we outline and test a
computationally feasible two-step procedure: In a first step,
mean-curvature motion is used for segmentation, yielding subvolumes
that contain with a high probability macromolecules in the expected
size range. Subsequently, the particles contained in the subvolumes are
identified by cross-correlation, using a set of three-dimensional
templates. With simulated and real tomographic data we demonstrate that
such an approach is feasible and we explore the detection limits. Even
structurally similar particles, such as the thermosome, GroEL, and the
20S proteasome can be identified with high fidelity. This opens up
exciting prospects for mapping the territorial distribution of
macromolecules and for analyzing molecular interactions in