Cannabinoids have been considered for some time as potent therapeutic agents in chronic pain management. Central and systemic administration of natural, synthetic and endogenous cannabinoids produce antinociceptive and antihyperalgesic effects in both acute and chronic animal pain models. Although much of the existing data suggest that the analgesic effects of cannabinoids are mediated via neuronal CB1 receptors, there is increasing evidence to support a role for peripheral CB2 receptors, which are expressed preferentially on immune cells. As yet, little is known about the central contribution of CB2 in neuropathic pain states. We report here that chronic pain models associated with peripheral nerve injury, but not peripheral inflammation, induce CB2 receptor expression in a highly restricted and specific manner within the lumbar spinal cord. Moreover, the appearance of CB2 expression coincides with the appearance of activated microglia.

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNA's are expressed at low levels in a variety of tissues.

Given the high homology in amino acid sequence between the ␦-opioid receptor and the two other types ( and ), distinct residues in this receptor may confer its selectivity to some ligands. In order to identify molecular determinants in the human ␦ receptor responsible for the selectivity of ␦-selective ligands, two different ␦/ chimeras were constructed. In the first one, the ␦ sequence from the top of transmembrane 5 to the C terminus was replaced by the equivalent sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the ␦ receptor were replaced by the counterpart. These two chimeras retained the ability to bind the nonselective bremazocine but completely lost the ability to bind different ␦-selective ligands. These results suggested that the region of the third extracellular loop of the ␦ receptor is crucial for the type selectivity. Opioid receptors are cell surface glycoproteins that constitute specific binding sites for a variety of compounds used for treating pain. Extensive pharmacological studies led to the definition of three opioid receptor types, , , and ␦ (1, 2). Indeed, the availability of highly selective ligands permitted a better characterization of these three types of receptors. In the past few years, cDNAs encoding the , , and ␦ of different species have been cloned (reviewed in Ref.3). However, human receptors represent the ultimate therapeutic targets. Thus, the cloning of human , , and ␦ cDNAs (4 -6) provided particularly relevant tools for opioid drug discovery.Analysis of the predicted amino acid sequences of the three human opioid receptors has shown that these receptors have characteristics common to the guanine nucleotide-binding regulatory protein-coupled receptors with an extracellular N-terminal domain, a cytoplasmic C-terminal domain, and seven putative transmembrane domains (7,8). Moreover, given their high degree of identity (approximately 60%) with the highest similarity in the transmembrane domains and intracellular loops, distinct residues in these receptors may confer their selectivity to some ligands. Binding sites of selective ligands most probably reside, at least in part, in the divergent regions that are the extracellular loops and the N-terminal domain. The C-terminal domain is also a divergent region, but its involvement in binding of ligands is unlikely.All of the opioid analgesics currently used clinically interact with the receptor and are known to induce severe side effects and addiction (9, 10). These major disadvantages were less pronounced with the use of a number of agonists selective for the receptor which also display analgesic properties (11-13), suggesting that some effective analgesics with minimal side effects, selectively interacting with one of the three types of opioid receptors, may be developed. Particularly, ␦ receptors that bind enkephalin-like peptides with high affinity have been proposed to mediate analgesia (14 -16) and to induce weak opiate physical dependence (17, 18), making them an interesting t...

The thyrotropin-releasing hormone (TRH) 1 is a tripeptide (pyroglutamic acid-histidine-proline-amide) synthesized from a precursor polypeptide whose sequence contains 5 copies of the TRH sequence (5-7).Originally isolated from the hypothalamus, TRH is present in the central nervous system (thalamus, cerebral cortex, and spinal cord,) as well as in the periphery (pancreas, gastrointestinal tracts, and placenta). In the hypothalamus, TRH is synthesized by peptidergic neurons of supraoptic and paraventricular nuclei. It is then axonally transported to be stored in the median eminence. When secreted in the bloodstream, it reaches the pituitary where it stimulates the production of thyroid stimulating hormone which in turn stimulates the production of thyroxin (T 4 ) in the thyroid gland (8).In addition to this pivotal role in controlling the synthesis and secretion of thyroid stimulating hormone and other hormones from the anterior pituitary, TRH has been implicated as a neurotransmitter (9). TRH abundantly exists in the central nervous system and exogenous administration of TRH elicits a variety of behavioral changes (see Ref. 10 for a review).The distribution of TRH containing cells, fibers, or receptors suggests a potential role of TRH in the perception of noxious stimuli. TRH is present in the periaqueductal gray, the nuclei raphe magnus and pallidus, and the dorsal horn of the spinal cord. TRH-binding sites have been described in the brain, the pituitary, in both the dorsal and ventral horns of the spinal cord as well as in peripheral tissues. When injected centrally, TRH induces a short lasting supraspinal antinociception. The analgesia induced by intracerebroventricular TRH injection is powerful since it is twice as great, on a molar basis, as that of morphine (11). This TRH-induced antinociception is detected in models of chemically and mechanically, but not thermally induced pain. On the other hand, intrathecal TRH injections do not affect basal antinociceptive thresholds (11). However, it is known that TRH enhances spinal reflexes (both in vivo and in vitro) and modulates pain transmission (4,12,13). Although the mode of action of TRH at the level of the spinal cord is unclear, there is evidence suggesting that the TRH-induced facilitation of spinal transmission involves the activation of the N-methyl-D-asparatate receptor (14).TRH actions are mediated by the stimulation of specific cell surface receptors. Studies of pituitary TRH receptors have suggested that the TRHR1 receptor triggers the phospholipase C-protein kinase C transduction pathway (3,15). A cDNA sequence encoding a G-protein-coupled TRHR was originally isolated from mouse pituitary using an expression cloning strategy (15). Subsequently, several groups have described the cloning of rat TRH receptor cDNAs expressed in a pituitary tumor cell line (GH 3 ) or in the pituitary gland (1, 3). In addition, two isoforms of the rat TRHR have been shown to be generated from a single gene by alternative splicing (2). These isoforms are 387 and 412 amin...

General-base catalysis in the active site of serine proteases is carried out by the imidazole side chain of a histidine. During formation of the transition state, an adjacent carboxylic acid group stabilizes the positive charge that forms on the general-base catalyst and as a result contributes several orders of magnitude to the catalytic efficiency of these enzymes. In the recently discovered family of self-cleaving proteins exemplified by the LexA repressor of Escherichia coli, instead of the imidazole of a histidine, the active-site general-base catalyst was found to be the epsilon-amino of a lysine. The considerably higher capacity of the lysine side chain for proton acceptance raises interesting questions concerning the role of electrostatic interactions in the mechanism of proton transfer by this highly basic group. The negative charge elimination studies described here and their effects on the kmax and pK of LexA self-cleavage are consistent with a model in which electrostatic interactions between an acidic side chain and the general-base catalyst form a barrier to proton transfer. The implications are that the epsilon-amino group, unlike the imidazole group, is capable of effecting proton transfer without the intervention of a countercharge.

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Two hybrid replicons containing polyomavirus (Py) genomes with large duplications of the viral late coding sequences were transfected into various permissive mouse cell lines. In all cell lines, either replicon yielded the sole amplifiable product expected from intramolecular homologous recombination, unit-length Py DNA (P155). In normal and in Py-transformed cells, such recombination was highly effective and involved sequences previously found to act as recombination hot spots (S repeats). In cells transformed by simian virus 40, however, these hot spots were inoperative in the generation of P155, which occurred with a reduced efficiency. These data confirm and extend earlier data indicating that the nature of products arising from recombination in Py replicons is tightly controlled by both cis- and trans-acting factors.

Next-generation sequencing (NGS) has revolutionized genetics and enabled the accurate identification of many genetic variants across many genomes. However, detection of biologically important low-frequency variants within genetically heterogeneous populations remains challenging, because they are difficult to distinguish from intrinsic NGS sequencing error rates. Approaches to overcome these limitations are essential to detect rare mutations in large cohorts, virus or microbial populations, mitochondria heteroplasmy, and other heterogeneous mixtures such as tumors. Modifications in library preparation can overcome some of these limitations, but are experimentally challenging and restricted to skilled biologists. This paper describes a novel quality filtering and base pruning pipeline, called Complex Heterogeneous Overlapped Paired-End Reads (CHOPER), designed to detect sequence variants in a complex population with high sequence similarity derived from All-Codon-Scanning (ACS) mutagenesis. A novel fast alignment algorithm, designed for the specified application, has O(n) time complexity. CHOPER was applied to a p53 cancer mutant reactivation study derived from ACS mutagenesis. Relative to error filtering based on Phred quality scores, CHOPER improved accuracy by about 13% while discarding only half as many bases. These results are a step toward extending the power of NGS to the analysis of genetically heterogeneous populations.