Targeting fatty acid amide hydrolase as a therapeutic strategy for antitussive therapy.

Cough is the most common reason to visit a primary care physician, yet it remains an unmet medical need. Fatty acid amide hydrolase (FAAH) is an enzyme that breaks down endocannabinoids, and inhibition of FAAH produces analgesic and anti-inflammatory effects. Cannabinoids inhibit vagal sensory nerve activation and the cough reflex, so it was hypothesised that FAAH inhibition would produce antitussive activity elevation of endocannabinoids.

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands.

Selective modulation of hepatic cytochrome P450 and flavin monooxygenase 3 expression during citrobacter rodentium infection in severe combined immune-deficient mice.

The profile of selective modulation of hepatic cytochrome P450 (P450) gene expression caused by infection with the murine intestinal pathogen Citrobacter rodentium has been well characterized in multiple genetic backgrounds; yet, the mechanisms underlying this modulation are still not entirely understood. Although several studies have addressed the roles of cytokines from the innate immune system, the influence of the adaptive immune system is not known. To address this deficiency, we used mice harboring the severe combined immune deficiency (SCID) spontaneous mutation, which lack mature T and B lymphocytes and are unable to mount an acquired immune response.

A Comparison of the α2/3/5 Selective Positive Allosteric Modulators L-838,417 and TPA023 in Preclinical Models of Inflammatory and Neuropathic Pain.

GABA(A) receptors containing α2/3 subunits are current targets in the battle to develop new pain medications, as they are expressed in the spinal cord where increasing inhibitory drive should result in analgesia. However, this approach is prone to a range of side effects including sedation, cognitive impairment, and abuse as a consequence of the widespread influence of GABA. The ability to make subtype selective low-efficacy benzodiazepine compounds, which potentiate the action of GABA at specific α subunits, has the potential to reduce this side effect profile.

Selective role for tumor necrosis factor-α, but not interleukin-1 or Kupffer cells, in down-regulation of CYP3A11 and CYP3A25 in livers of mice infected with a noninvasive intestinal pathogen.

Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1-/-), interleukin-1 (IL1) receptor null mice (IL1R1-/-), and Kupffer cell depleted mice with C.

Understanding the effect of different assay formats on agonist parameters: a study using the µ-opioid receptor.

The correct interpretation of data is fundamental to the study of G-protein-coupled receptor pharmacology. Often, new assay technologies are assimilated into the drug discovery environment without full consideration of the data generated. In this study, the authors look at µ-opioid receptor agonists in three different assays: (1) [(35)S]GTPγS binding, (2) inhibition of forskolin-stimulated cAMP production, and (3) β-arrestin recruitment.

Stabilization and spectroscopic characterization of the dioxygen complex of wild-type cytochrome P4502B4 (CYP2B4) and its distal side E301Q, T302A and proximal side F429H mutants at subzero temperatures.

Mammalian cytochrome P450 2B4 (CYP2B4) is a phenobarbital-inducible rabbit hepatic monooxygenase that catalyzes the N-demethylation of benzphetamine and metabolism of numerous other compounds. To probe the interactions of the heme environment and bound benzphetamine with the dioxygen (O₂) complex of CYP2B4, homogeneous O₂ complexes of the wild-type enzyme and three mutants at sites of conserved amino acids, two on the heme distal side (T302A and E301Q) and one on the proximal side (F429H), have been prepared and stabilized at ~-50°C in mixed solvents (60-70% v/v glycerol). We report that the magnetic circular dichroism and electronic absorption spectra of wild-type oxyferrous CYP2B4, in the presence and absence of substrate, are quite similar to those of the dioxygen complex of bacterial cytochrome P450-CAM (CYP101).

Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS).

Nitric oxide synthase (NOS) catalyzes the NADPH- and O(2)-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an N(G)-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV-Vis absorption spectroscopic techniques.

2,4-Diaminopyridine delta-opioid receptor agonists and their associated hERG pharmacology.

A number of libraries were produced to explore the potential of 2,4-diaminopyridine lead 1. The resulting diaminopyridines proved to be potent and selective delta-opioid receptor agonists. Several rounds of lead optimisation using library chemistry identified compound 17 which went on to show efficacy in an electromyography model of neuropathic pain.

TLR4-dependent and -independent regulation of hepatic cytochrome P450 in mice with chemically induced inflammatory bowel disease.

The transcription and protein expression of many cytochrome P450 (P450) genes are down-regulated in animal models of inflammation and infection. We determined previously that hepatic P450 mRNAs are selectively regulated in a mouse model of enteropathogenic bacterial infection, and that this regulation was not dependent on the lipopolysaccharide (LPS) receptor protein toll-like receptor 4 (TLR4). In the dextran sulfate sodium (DSS) model of chemically induced inflammatory bowel disease (IBD), the reduction in activities of several hepatic P450 enzymes were concluded to be partially dependent on LPS from commensal bacteria [Masubuchi Y, Horie T.

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals.

The effect of deletion of the orphan G - protein coupled receptor (GPCR) gene MrgE on pain-like behaviours in mice.

Background: The orphan GPCR MrgE is one of an extended family of GPCRs that are expressed in dorsal root ganglia (DRG). Based on these expression patterns it has been suggested that GPCRs like MrgE may play a role in nociception however, to date, no direct supporting evidence has emerged. We generated mutant mice lacking MrgE and examined the effects of deletion of this gene in three pain behavioural models.

[3H] pregabalin binding is increased in ipsilateral dorsal horn following chronic constriction injury.

Pregabalin, a 3-substituted analogue of gamma-amino butyric acid has recently been approved for treatment of neuropathic pain. We have investigated the anatomical binding profile of [(3)H] pregabalin following chronic constriction injury (CCI) and compared this with alpha 2 delta 1 subunit expression using in situ hybridisation. We report here that the intensity and distribution pattern of [(3)H] pregabalin binding is altered in the ipsilateral dorsal horn following CCI and this is associated with a corresponding increase in alpha 2 delta 1 mRNA in the ipsilateral dorsal root ganglion (DRG).

Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin.

Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain.

Evaluation of electron-withdrawing group effects on heme binding in designed proteins: implications for heme a in cytochrome c oxidase.

Heme a, the metalloporphyrin cofactor unique to cytochrome c oxidases, differs from the more common heme b by two chemical modifications, a C-2 hydroxyethylfarnesyl group and a C-8 formyl group. To elucidate a role of the C-8 formyl group, we compare the heme affinity, spectroscopy, and electrochemistry of a heme a mimic, Fe(diacetyldeuterioporphyrin IX) or Fe(DADPIX), with heme b, Fe(protoporphryrin IX) or Fe(PPIX), incorporated into a designed heme protein. The [Delta7-H3m]2 protein ligand, or maquette, selected for this study contains two equivalent bis-(3-methyl-L-histidine) heme binding sites within a four-alpha-helix bundle scaffold.

The human FK506-binding proteins: characterization of human FKBP19.

Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues.

New targets for neuropathic pain therapeutics.

Neuropathic pain (NeP) is initiated by a lesion or dysfunction in the nervous system. Unlike physiological pain it serves no useful purpose and is usually sustained and chronic. NeP encompasses a wide range of pain syndromes of diverse aetiologies which together account for > 12 million sufferers in the US.

Design of a five-coordinate heme protein maquette: a spectroscopic model of deoxymyoglobin.

The substitution of 1-methyl-l-histidine for the histidine heme ligands in a de novo designed four-alpha-helix bundle scaffold results in conversion of a six-coordinate cytochrome maquette into a self-assembled five-coordinate mono-(1-methyl-histidine)-ligated heme as an initial maquette for the dioxygen carrier protein myoglobin. UV-vis, magnetic circular dichroism, and resonance Raman spectroscopies demonstrate the presence of five-coordinate mono-(1-methyl-histidine) ligated ferrous heme spectroscopically similar to deoxymyoglobin. Thermodynamic analysis of the ferric and ferrous heme dissociation constants indicates greater destabilization of the ferric state than the ferrous state.

Same-day discharge is preferred by the majority of the patients undergoing radial PCI.

There is limited data on patient preference for same-day discharge PCI. We contacted 953 patients who had same-day discharge radial PCI between 1998 and 2001 and checked whether they were satisfied with same-day discharge and whether they had any complications within 30 days post-PCI (vascular, repeat angiogram/PCI). Complications and health status were also verified by checking hospital records, our province-wide cath lab database and provincial vital statistics, as well as by contacting the referring doctor.

Comparison of the radial and femoral approaches in left main PCI: a retrospective study.

Transradial percutaneous coronary intervention (PCI) is a safe and effective method of percutaneous revascularization. However, there are no data on the efficacy of the transradial approach in left main (LM) PCI. We studied 80 patients (pts) who underwent LM PCI between February 1994 and January 2002, and compared the radial (27 pts) and femoral (53 pts) approaches.

Metalloproteinase shedding of Fas ligand regulates beta-amyloid neurotoxicity.

Extracellular deposits of beta-amyloid (Abeta) peptide closely match areas of neuronal loss in, and are a postmortem diagnostic indicator of, Alzheimer's disease. Neuronal cultures treated with fibrillar Abeta can be protected from neurotoxicity by caspase-8 inhibition or the expression of dominant-negative FADD, both of which are components of the Fas death receptor pathway, and neurons with defective Fas and FasL are resistant to Abeta neurotoxicity. The receptor binding region of FasL can be shed from cells by metalloproteinases, and this process greatly reduces its proapoptotic activity.

Mitogen-activated protein kinase kinase 7 is activated during low potassium-induced apoptosis in rat cerebellar granule neurons.

A stress-activated protein kinase pathway comprising mitogen-activated protein kinase kinases (MKKs), c-Jun N-terminal kinase (JNK) and the transcription factor c-Jun is implicated in neuronal apoptosis. Using an immune-complex kinase assay, we measured the activation of MKK4 and MKK7 in low potassium (LK)-induced apoptosis of rat cerebellar granule neurons (CGN). MKK7, but not MKK4, was activated within the first 4-6 h in four independent sets of 14-h CGN apoptosis time-courses.

Management of Root-knot and Reniform Nematodes in Ultra-Narrow Row Cotton with 1,3-Dichloropropene.

Ultra-narrow row cotton studies were conducted during 1999 at two field sites in northern Florida. One site was naturally infested with Meloidogyne incognita Race 3 and the other with Rotylenchulus reniformis. The fumigant 1,3-dichloropropene (1,3-D) was applied broadcast at rates of 0, 16, 32, 48, 64, 80, and 96 kg ai.

Overwinter Population Dynamics of Heterodera glycines.

The purpose of this research was to compare the overwinter survival of populations of Heterodera glycines at different latitudes in the United States and the effect of changing latitudes before and after the initiation of dormancy. Soil samples infested with H. glycines were collected in August or October in 1992 to 1994 from soybean fields in two to four states (combinations of Arkansas, Florida, Minnesota, Missouri, and Wisconsin).