Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes.

CXCR7/CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration.

G protein-coupled receptor hetero-oligomerization is emerging as an important regulator of ligand-dependent transmembrane signaling, but precisely how receptor heteromers affect receptor pharmacology remains largely unknown. In this study, we have attempted to identify the functional significance of the heteromeric complex between CXCR4 and CXCR7 chemokine receptors. We demonstrate that co-expression of CXCR7 with CXCR4 results in constitutive recruitment of β-arrestin to the CXCR4·CXCR7 complex and simultaneous impairment of G(i)-mediated signaling.

Cell surface targeting of mu-delta opioid receptor heterodimers by RTP4.

Mu opioid receptors are G protein-coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that mu-delta opioid heterodimers have a pharmacologic profile distinct from those of the mu or delta homodimers. Because the heterodimers exhibit distinct signaling properties, the protein and mechanism regulating their levels have significant effects on morphine-mediated physiology.

Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies.

The majority of studies examining activity-induced conformational changes in G protein-coupled receptors have focused on transmembrane helices or intracellular regions. Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process. To begin to address this, we generated a series of antibodies to the N-terminal region of opioid receptors.

Conformation state-sensitive antibodies to G-protein-coupled receptors.

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors.

Peptidomics of Cpefat/fat mouse hypothalamus and striatum: effect of chronic morphine administration.

Chronic morphine administration is known to affect several neuropeptide systems, and this could contribute to the behavioral effects of opiates. To quantitate global changes in neuropeptide levels upon chronic morphine administration, we took advantage of a method that allows selective isolation of neuropeptides from brains of mice lacking carboxypeptidase E (Cpefat/fat mice), a critical enzyme in the generation of many neuroendocrine peptides. We used a differential labeling procedure with stable isotopic tags and mass spectrometry to quantitate the relative changes in a number of hypothalamic and striatal peptides in Cpefat/fat mice chronically treated with morphine.

Targeting opioid receptor heterodimers: strategies for screening and drug development.

G-protein-coupled receptors are a major target for the development of new marketable drugs. A growing number of studies have shown that these receptors could bind to their ligands, signal, and be internalized as dimers. Most of the evidence comes from in vitro studies, but recent studies using animal models support an important role for dimerization in vivo and in human pathologies.

Inverse agonism and neutral antagonism at wild-type and constitutively active mutant delta opioid receptors.

The delta opioid receptor modulates nociceptive and emotional behaviors. This receptor has been shown to exhibit measurable spontaneous activity. Progress in understanding the biological relevance of this activity has been slow, partly due to limited characterization of compounds with intrinsic negative activity.

Opioid receptor random mutagenesis reveals a mechanism for G protein-coupled receptor activation.

The high resolution structure of rhodopsin has greatly enhanced current understanding of G protein-coupled receptor (GPCR) structure in the off-state, but the activation process remains to be clarified. We investigated molecular mechanisms of delta-opioid receptor activation without a preconceived structural hypothesis. Using random mutagenesis of the entire receptor, we identified 30 activating point mutations.

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling.

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities.

[Impact of initial antibiotic therapy on the course of resistance to fluoroquinolones and aminoglycosides in Gram-negative bacilli isolated from intensive care patients].

Objective: To evaluate the effect of the initial antibiotic therapy associating a betalactam antibiotic (BLA) with either an aminoglycoside (AG) or a fluoroquinolone (FQ) on the development of resistance of gram-negative bacilli in an intensive care unit.

Study Design: Prospective bacteriological surveillance study.

Patients: The study included 51 patients experiencing a second infection with gram-negative organisms, eight days or more after a first infection.

Haemodynamic changes induced by two I:E ratios: a transoesophageal echocardiographic study.

Purpose: To assess the effects of controlled ventilation with two I:E ratios on haemodynamic and left ventricular function in mechanically ventilated patients with moderate to severe respiratory disease, using fluctuation of the arterial pressure waveform and the changes in left ventricular areas obtained by transoesophageal echocardiography.

Methods: Nine patients had their lungs ventilated using volume controlled ventilation with two I:E ratios 1:3 and 1:1). Respiratory rate was adjusted so that six cardiac beats occurred during a respiratory cycle.