4,236 research outputs found
A clickable analogue of ketamine retains NMDA receptor activity, psychoactivity, and accumulates in neurons
Ketamine is a psychotomimetic and antidepressant drug. Although antagonism of cell-surface NMDA receptors (NMDARs) may trigger ketamineâs psychoactive effects, ketamine or its major metabolite norketamine could act intracellularly to produce some behavioral effects. To explore the viability of this latter hypothesis, we examined intracellular accumulation of novel visualizable analogues of ketamine/norketamine. We introduced an alkyne âclickâ handle into norketamine (alkyne-norketamine, A-NK) at the key nitrogen atom. Ketamine, norketamine, and A-NK, but not A-NK-amide, showed acute and persisting psychoactive effects in mice. This psychoactivity profile paralleled activity of the compounds as NMDAR channel blockers; A-NK-amide was inactive at NMDARs, and norketamine and A-NK were active but ~4-fold less potent than ketamine. We incubated rat hippocampal cells with 10âÎŒM A-NK or A-NK-amide then performed Cu(2+) catalyzed cycloaddition of azide-Alexa Fluor 488, which covalently attaches the fluorophore to the alkyne moiety in the compounds. Fluorescent imaging revealed intracellular localization of A-NK but weak A-NK-amide labeling. Accumulation was not dependent on membrane potential, NMDAR expression, or NMDAR activity. Overall, the approach revealed a correlation among NMDAR activity, intracellular accumulation/retention, and behavioral effects. Thus, we advance first generation chemical biology tools to aid in the identification of ketamine targets
Multiple functional neurosteroid binding sites on GABAA receptors
Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1ÎČ3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and ÎČ3 (labeled residue ÎČ3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues ÎČ3-L294 and G308) in the interface between the ÎČ3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and ÎČ3-α1 intersubunit sites are critical for neurosteroid action
Site-specific effects of neurosteroids on GABA(A) receptor activation and desensitization
This study examines how site-specific binding to three identified neurosteroid-binding sites in the
Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications
We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Ă
resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 Ă 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications
Search for the lepton-flavor-violating decays Bs0âe±Όâ and B0âe±Όâ
A search for the lepton-flavor-violating decays Bs0âe±Όâ and B0âe±Όâ is performed with a data sample, corresponding to an integrated luminosity of 1.0ââfb-1 of pp collisions at âs=7ââTeV, collected by the LHCb experiment. The observed number of Bs0âe±Όâ and B0âe±Όâ candidates is consistent with background expectations. Upper limits on the branching fractions of both decays are determined to be B(Bs0âe±Όâ)101ââTeV/c2 and MLQ(B0âe±Όâ)>126ââTeV/c2 at 95% C.L., and are a factor of 2 higher than the previous bounds
Observation of the decay
The decay is observed for the first
time, using proton-proton collisions collected with the LHCb detector
corresponding to an integrated luminosity of 3fb. A signal yield of
decays is reported with a significance of 6.2 standard deviations.
The ratio of the branching fraction of \B_c \rightarrow J/\psi K^+ K^- \pi^+
decays to that of decays is measured to be
, where the first uncertainty is statistical and the
second is systematic.Comment: 18 pages, 2 figure
Observation of two new baryon resonances
Two structures are observed close to the kinematic threshold in the mass spectrum in a sample of proton-proton collision data, corresponding
to an integrated luminosity of 3.0 fb recorded by the LHCb experiment.
In the quark model, two baryonic resonances with quark content are
expected in this mass region: the spin-parity and
states, denoted and .
Interpreting the structures as these resonances, we measure the mass
differences and the width of the heavier state to be
MeV,
MeV,
MeV, where the first and second
uncertainties are statistical and systematic, respectively. The width of the
lighter state is consistent with zero, and we place an upper limit of
MeV at 95% confidence level. Relative
production rates of these states are also reported.Comment: 17 pages, 2 figure
Observation of associated production of a boson with a meson in the~forward region
A search for associated production of a boson with an open charm meson is
presented using a data sample, corresponding to an integrated luminosity of
of proton--proton collisions at a centre-of-mass energy
of 7\,TeV, collected by the LHCb experiment. %% Seven candidate events for
associated production of a boson with a meson and four candidate
events for a boson with a meson are observed with a combined
significance of 5.1standard deviations. The production cross-sections in the
forward region are measured to be where the first uncertainty is statistical and the
second systematic.Comment: 18 pages, 2 figure
Observations of BÂșsâÏ(2S)η and BÂș(s)âÏ(2S)Ï+Ï- decays
First observations of the B0s
âÏ(2S)η, B0 âÏ(2S)Ï
+
Ï
â and B0s
âÏ(2S)Ï
+
Ï
â decays are made
using a dataset corresponding to an integrated luminosity of 1.0 fbâ1 collected by the LHCb experiment in
protonâproton collisions at a centre-of-mass energy of
â
s = 7 TeV. The ratios of the branching fractions
of each of the Ï(2S) modes with respect to the corresponding J/Ï decays are
B(B0s
âÏ(2S)η)
Ă·
B(B0s
âJ/Ïη)
= 0.83± 0.14 (stat)±0.12 (syst) ±0.02 (B),
;
B(B0âÏ(2S)Ï
+
Ï
â
)
Ă·
B(B0âJ/ÏÏ
+
Ï
â
)
= 0.56± 0.07 (stat)±0.05 (syst)± 0.01 (B),
;
B(B0s
âÏ(2S)Ï
+
Ï
â
)
Ă·
B(B0s
âJ/ÏÏ
+
Ï
â
)
= 0.34± 0.04 (stat)±0.03 (syst)± 0.01 (B),
where the third uncertainty corresponds to the uncertainties of the dilepton branching fractions of the J/Ï
and Ï(2S) meson decays
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