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Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains., The extracellular region of Smoothened (SMO) is composed of an N-terminal CRD followed by a small linker domain, which then connects to the TMD and a C-terminal intracellular domain (ICD; [...]

Type-A γ-aminobutyric acid receptors ([GABA.sub.A]Rs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in [GABA.sub.A]R signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a [GABA.sub.A]R, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of [GABA.sub.A]R modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission., In response to binding the neurotransmitter GABA, released at inhibitory synapses, [GABA.sub.A]R chloride channels open and depress neuronal excitability in the adult central nervous system (1). [GABA.sub.A]Rs belong to a [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2009.12.005 Byline: Paul S. Miller, Trevor G. Smart Abstract: It is over forty years since the major neurotransmitters and their protein receptors were identified, and over twenty years since determination of the first amino-acid sequences of the Cys-loop receptors that recognize acetylcholine, serotonin, GABA and glycine. The last decade has seen the first structures of these proteins (and related bacterial and molluscan homologues) determined to atomic resolution. Hopefully over the next decade, more detailed molecular structures of entire Cys-loop receptors in drug-bound and drug-free conformations will become available. These, together with functional studies, will provide a clear picture of how these receptors participate in neurotransmission and how structural variations between receptor subtypes impart their unique characteristics. This insight should facilitate the design of novel and improved therapeutics to treat neurological disorders. This review considers our current understanding about the processes of agonist binding, receptor activation and channel opening, as well as allosteric modulation of the Cys-loop receptor family. Author Affiliation: Department of Neuroscience, Physiology & Pharmacology, University College London, Gower Street, London WC1E 6BT, UK

This study was conducted to evaluate the overall performance of the Precision Agricultural-Landscape Modeling System (PALMS) for calculating runoff and soil loss under cropped conditions. The PALMS model uses a lognormal distribution of saturated hydraulic conductivity across the fields to simulate typical soil heterogeneity within soil texture classes. Runoff and soil loss data were collected in three farm fields for a total of 75 runoff events during 2 yr under six cropping scenarios (alfalfa [Medicago sativa L.] and corn [Zea mays L.] no-tilled, corn and soybean [Glycine max (L.) Merr.] moldboard plowed, and alfalfa and corn chisel plowed). For individual storms, calculated runoff and sediment loss from PALMS were compared with corresponding measurements for each farm during the entire cropping season. The coefficient of determination ([r.sup.2]) between runoff calculations and measurements was 0.84. The [r.sup.2] between soil loss calculations and measurements with the storm-by-storm simulations during both seasons was 0.78. Based on these continuous simulation results, the PALMS calculations appear to have lower relative errors with large events than small events, a desirable result because large events are most important in assessing the environmental consequences of management practices. Abbreviations: DEM, digital elevation model; EPIC, Erosion Productivity Impact Calculator; ME, model efficiency; PALMS, Precision Agricultural-Landscape Modeling System; RUSLE, Revised Universal Soil Loss Equation; USLE, Universal Soil Loss Equation; WEPP, Water Erosion Prediction Project

The X-ray structure of a DNA duplex containing a synthetic [N.sup.4]C-ethyl-[N.sup.4]C interstrand cross-link between the cytosines of the central CpG step is determined at 1.65 Angstrom resolution. This structure reveals that the ethyl cross-link in the CpG major grooves does not significantly disrupt the B-form DNA helix.

Abstract: Polypyrimidine oligonucleotides can bind to tracts of contiguous purines in double-stranded DNA to form triple-stranded complexes. The stability of the triplex is reduced significantly if the target purine tract [...]

Several antitumour drugs react with DNA to form interstrand cross-links, causing chromosomal alterations like DNA strand breakage and hyper-recombination on the DNA helix, necessitating repair of damage on both strands of the DNA. Two basic processes by which interstrand cross-link lesions are repaired, an error-free process and an error-prone process, rely on proteins in the nucleotide incision repair, homologous recombination and translesion DNA synthesis pathways.

The structural properties underpinning the differences in the immediate environment of a N(super 3)T-ethyl-N(super 3)T interstrand cross link that is inserted into a scaffold wherein the cross link is mispair-aligned is described. Results demonstrate that the lesion defined by mismatch-aligned minor groove N(super 3)T-ethyl-N(super 3)T cross-linked thymine bases produces very localized distortions in a DNA stem that may be difficult to recognize by repair mechanisms that are not transcription-or-replication-coupled.

A cross-link that bridges the N(super 3) positions of a mismatched thymide base pair is synthesized, as a part of the studies on DNA duplexes that contain alkyl interstrand cross-links. Fully cross-linked duplexes were synthesized using an orthogonal approach to selectively remove protecting groups that allowed construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis.

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, β-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αβ, α4βδ, α6βδ and α5βγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1βγ, α2βγ and α3βγ receptor responses5,7–12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αβ GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA–Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αβ receptors that adapt them to a role in tonic signalling.Cryo-electron microscopy structures are used to identify mechanisms underlying distinct features of extrasynaptic type A γ-aminobutyric acid receptors. [ABSTRACT FROM AUTHOR]

The doctrine of unconscionable contracts is an equity doctrine. As such, it does not ask whether an agreement is or is not an enforceable contract; legality has no bearing. Rather, the doctrine asks whether it would be it be fit and proper . . . would it be fair . . . for a court to enforce such a contract. Using the doctrine, courts examine whether the price paid for a good or service far outstrips any benefit gained. What if that price is the surrender of a basic right, the Right (Not) to Associate? The Supreme Court says that a violation of that right is "always demeaning." Would that be a price, to quote Lord Chancellor Hardwicke, "such as no man in his senses would make, or as no honest man would come into"? This Article is my latest collateral attack on Citizens United. The main ideas discussed are unconscionable contracts and the Right (Not) to Associate. A little bit of time is spent on the particulars of Delaware corporate law regarding the owner of record and the Employee Retirement Income Security Act (ERISA). Somehow, it ends up being about the unfairness of giving private corporations, and the officers that rule them, an exalted place in our political system. [ABSTRACT FROM AUTHOR]

Results show that the cross-link N(sup)4C-Ethyl-N(sup)4C interstrand in the undecamer deoxyoligonucleotide imitates the BCNU-induced G-C cross-link and the C-C cross-link, rendering this DNA duplex recognizable by different DNA repair mechanisms.

Results indicate that bending deformations in the helix involving the -CNG- sequence cross-linked DNA duplexes may be controlled by varying the cross-link length. Data show that the cross-link containing 2,4,or 7 methylene groups joins the C residues of a -CNG- sequence in the DnA duplex.

A threshold level of the triple helix forming oligonucleotides (TFOs), consisting of three or four 2'-AE residues, are 10-fold more active than those with just 2 2'-AE residues in a hypoxanthine phosphoribosyltransferase (HPRT) activity monitored gene knockout assays. Further, the frequencies of HPRT activity with the active TFOs are 300-400 fold higher than the background activity.

This articles describes the synthesis of N(sup)4C--ethyl--N(sup)4C interstrand cross-linked DNA duplexes employing N(sup)4C--ethyl--N(sup)4C phosphoramidite reagent. The ligated oligomers containing staggered cross-links exhibit differences in structure and stability with -GC- staggered cross-links disrupt hydrogen bonding and affecting helix stability by perturbing adjacent base pairs.

DNA duplexes with N4C-ethyl-N4C interstrand cross-links were synthesized.

Psoralen-conjugated chimeric oligonucleoside methylphosphonates and their triplex formation are discussed. Oligomers that are made up of pyrimidine nucleosides form stable triplexes at neutral pH. Triplex-forming oligonucleotides (TFOs) have been thought to perhaps be DNA-specific ligands that may be used for controlling gene expression. TFO here refers to oligonucleotides designed to bind to purine bases in the major groove of the DNA target.

Oligonucleotides have been synthesized that can bind to TAR RNA and are resistant to nuclease degradation. TAR RNA regulates HIV replication and these oligonucleotides could be used as antisense drugs to block viral replication.

The interaction between the antisense activity and oligomer binding affinity in cell cultures suggests that oligo-2'-O-methylribonucleoside methylphosphonates (mr-OMPs) can be used as effective antisense reagents. The capacity of mr-OMP to bind to its RNA target is higher than that of oligo-2'-deoxyribonucleoside methyphosphonate. The mr-OMP also inhibits herpes simplex virus type 1 (HSV-1) replication in HSV-1 infected Vero cells in culture.

Melting experiments on psoralen-derivatized oligonucleoside methylphosphonates with a random coil or a hairpin in solution reveal that the binding and cross-linking reactions are affected by target structure and sequence. The oligomer binding site position in the target RNA and the nature of the oligomer backbone enhance the binding of oligonucleotides containing phosphodiester or phosphorothiate internucleotide linkages to RNA-hairpins.