Ring flexibility within tricyclic antidepressant drugs

The internal flexibility of the central seven-membered ring of a series of tricyclic antidepressant drugs (TCAs), imipramine {l}, amitriptyline {2}, doxepin {3}, and dothiepin {4}, has been investigated by H-1 and C-13 nuclear magnetic (NMR) techniques. Two dynamic processes were examined: ring inversion and bridge flexing. H-1 NMR lineshape analysis was used to obtain ring inversion barriers for 2-4. These studies yielded energy barriers of 14.3, 16.7, and 15.7 +/- 0.6 kcal/mol for the hydrochloride salts of doxepin, dothiepin, and amitriptyline, respectively. The barriers for the corresponding free bases were lower by 0.6 kcal/mol on average. (CT1)-C-13 relaxation measurements were used to determine the degree of bridge flexing associated with the central seven-membered ring for all four compounds. By fitting the T-1 data to a two-state jump model, lifetimes and amplitudes of rapid bridge flexing motions were determined. The results show that imipramine has the fastest rate of bridge flexing, followed by amitriptyline, doxepin, and dothiepin. The pharmacological profiles of the TCAs are complex and they interact with many receptor sites, resulting in numerous side effects and a general lack of understanding of their precise mode of action in different anxiety-related disorders. They all have similar three-dimensional structures, which makes it difficult to rationalize their differing relative potency in different assays/clinical settings. However, the clear finding here that there are significantly different degrees of internal mobility suggests that molecular dynamics should be an additional factor considered when trying to understand the mode of action of this clinically important family of molecules. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:713-721, 2001.

Activation of the peroxisome proliferator-activated receptor-alpha enhances cell death in cultured cerebellar granule cells

Peroxisome proliferator-activated receptor-alpha (PPAR alpha) is a member of the steroid hormone receptor superfamily. In rodents, PPAR alpha. alters genes involved in cell cycle regulation in hepatocytes. Some of these genes are implicated in neuronal cell death. Therefore, in this study, we examined the toxicological consequence of PPAR alpha activation in rat primary cultures of cerebellar granule neurons. Our studies demonstrated the presence of PPAR alpha mRNA in cultures by reverse transcriptase-polymerase chain reaction. After 10 days in vitro, cerebellar granule neuron cultures were incubated with the selective PPAR alpha activator 4-chloro-6-(2,3-xylidino)2-pyrimidinylthioacetic acid (Wy-14,643). The inherent toxicity of Wy-14,643 and the effect of PPAR alpha activation following toxic stimuli were assessed. In these studies, neurotoxicity was induced through reduction of extracellular [KCl] from 25 mM to 5.36 mM. We observed no inherent toxicity of Wy-1 4,643 (24 hr) in cultured cerebellar granule cells. However, after reduction of [KCl], cerebellar granule cell cultures incubated with Wy-14,643 showed significantly greater toxicity than controls. These results suggest a posssible role for PPAR(x in augmentation of cerebellar granule neuronal death after toxic stimuli. (C) 2001 Wiley-Liss, Inc.

Regulation of LIF receptor expression in vascular smooth muscle

Previous studies in our laboratory have shown that the pleiotropic cytokine leukemia inhibitory factor (LIF) inhibits neointimal formation and the development and progression of atherosclerotic and restenotic lesions in a rabbit model of disease. The present study demonstrates an upregulation of both the LIF receptor (LIFR)-α subunit and the signal transducing subunit gp130 following endothelial denudation of the carotid artery by balloon catheter. Continuous infusion of LIF (30 μg/kg/day) resulted in the downregulation of LIFR-a in injured arteries in vivo. Similarly, smooth muscle cells in vitro treated with LIF exhibited a time-dependent reduction in LIFR-a protein expression and the subsequent reduction in transcription of the TIMP-1 gene. However, in the presence of an intact endothelium, LIFR-a was upregulated in response to LIF, and accordingly the downstream induction of iNOS expression was also increased. Thus, LIF exerts more potent antiatherogenic effects in the vasculature when the endothelium is intact.

Immune-derived opioids and peripheral antinociception

1. Recent findings have suggested a significant involvement of the immune system in the control of pain. Immune cells contain opioid peptides that are released within inflamed tissue and act at opioid receptors on peripheral sensory nerve endings. It is also apparent that different types of lymphocytes contain P-endorphin, memory T cells containing more beta -endorphin than naive cells. 2. These findings highlight an integral link between immune cell migration and inflammatory pain, The present review highlights immune system involvement in the site-directed control of inflammatory pain. 3. Full-length mRNA transcripts for opioid precursor proteins are expressed in immune cells. Increased expression of pro-opiomelanocortin mRNA and beta -endorphin has been demonstrated in stimulated lymphocytes and lymphocytes from animals with inflammation. 4. Cytokines and corticotropin-releasing factor (CRF) release opioids from immune cells, Potent peripheral analgesia due to direct injection of CRF can be blocked by antagonists to CRF, antibodies to opioid peptides, antisense to CRF and opioid receptor-specific antagonists. The release of opioid peptides from lymphocytes is calcium dependent and opioid receptor specific. Furthermore, endogenous sources of opioid peptides produce potent analgesia when implanted into the spinal cord. 5. Activated immune cells migrate directly to inflamed tissue using cell adhesion molecules to adhere to the epithelial surface of the vasculature in inflamed tissue. Lymphocytes that have been activated can express opioid peptides, Memory type T cells that contain opioid peptides are present within inflamed tissue; naive cells are not present in inflamed tissue and do not contain opioid peptides, Inhibiting the migration of memory type T cells into inflamed tissue by blocking selectins results in reduced numbers of beta -endorphin containing cells, a reduced quantity of beta -endorphin in inflamed paws and reduced stress- and CRF-induced peripheral analgesia. 6. Immunosuppression is associated with increased pain in patients. Moreover, immunosuppression results in decreased lymphocyte numbers as well as decreased analgesia in animal models.

Growth hormone receptor and IGF-1 receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-1). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corti ticosteroid-treated group (N = 6) was administered prednisolone ( 1 mg/kg,) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion. no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

The human melanocortin-1 receptor locus: analysis of transcription unit, locus polymorphism and haplotype evolution

The complete sequence of the MCIR locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the NULP1 and TUBB4 genes. The immediate promoter region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF), however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5' promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and MCIR coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought. (C) 2001 Published by Elsevier Science B.V.

EphB2 and two of its ligands have dynamic protein expression patterns in the developing olfactory system

Primary olfactory neurons are located in the olfactory neuroepithelium lining the nasal cavity. Their axons converge and form glomeruli with the dendrites of second-order neurons in the olfactory bulb. The molecular basis of primary olfactory axon guidance, targeting and subsequent arborisation is largely unknown. In this study we examined the spatio-temporal expression of the Eph receptor EphB2 and its ligands, ephrin-B1 and ephrin-B2, during development of the rat primary olfactory system. Unlike in other regions of the nervous system where receptor and ligand expression patterns are usually non-overlapping, EphB2, ephrin-B1 and ephrin-B2 were all expressed by primary and second-order olfactory neurons. In the embryonic animal we found that these three proteins had distinct and different expression patterns. EphB2 was first expressed at E18.5 by the perikarya of primary olfactory neurons. In contrast, ephrin-B1 was expressed from E13.5 and was localised to the axons of these cells up to E18.5 but was then restricted to the perikarya. Ephrin-B2, however, was expressed by olfactory ensheathing cells. EphB2, ephrin-B1 and ephrin-B2 were also expressed in the prenatal olfactory bulb and were restricted to the perikarya of mitral cells. In the post-natal olfactory bulb there was a shift in the localisation of both EphB2 and ephrin-B1 to the dendritic arborisations of mitral cells. The dynamic and tightly regulated spatio-temporal expression patterns of EphB2, ephrin-B1 and ephrin-B2 by specific olfactory cell populations suggest that these molecules have the potential to regulate important developmental events in the olfactory system. (C) 2001 Elsevier Science B.V. All rights reserved.

Chemically and morphologically identifiable glomeruli in the rat olfactory bulb

Primary olfactory neurons that express the same odorant receptor are distributed mosaically throughout the olfactory neuroepithelium lining the nasal cavity, yet their axons converge and form discrete glomeruli in the olfactory bulb. We previously proposed that cell surface carbohydrates mediate the sorting out and selective fasciculation of primary olfactory axons en route to glomeruli. If this were the case, then axons that terminate in the same glomerulus would express the same complement of cell surface carbohydrates. In this study, we examined the expression of a novel carbohydrate (NOC-3) on neural cell adhesion molecule in the adult rat olfactory system. NOC-3 was expressed by a subset of neurons distributed throughout the olfactory neuroepithelium. The axons of these neurons entered the nerve fiber layer and terminated in a subset of glomeruli. It is interesting to note that we identified three unusually large glomeruli in the lateral, ventrolateral, and ventromedial olfactory bulb that were innervated by axons expressing NOC-3. NOC-3-expressing axons sorted out and fasciculated into discrete fascicles prior to entering these glomeruli. Each of these glomeruli was in a topographically fixed position in the olfactory bulbs of the same animal as well as in different animals, and their lengths were approximately 10% of the total length of the bulb. They could be identified reliably by both their topographical position and their unique morphology. These results reveal that axons expressing the same cell surface carbohydrates consistently target the same topographically fixed glomeruli, which supports a role for these molecules in axon navigation in the primary olfactory nerve pathway. J. Comp. Neurol. 436: 497-507, 2001. (C) 2001 Wiley-Liss, Inc.

Expression of glycoproteins in the vomeronasal organ reveals a novel spatiotemporal pattern of sensory neurone maturation

The main olfactory and the accessory olfactory systems are both anatomically and functionally distinct chemosensory systems. The primary sensory neurones of the accessory olfactory system are sequestered in the vomeronasal organ (VNO), where they express pheromone receptors, which are unrelated to the odorant receptors expressed in the principal nasal cavity. We have identified a 240 kDa glycoprotein (VNO240) that is selectively expressed by sensory neurones in the VNO but not in the main olfactory neuroepithelium of mouse. VNO240 is first expressed at embryonic day 20.5 by a small subpopulation of sensory neurones residing within the central region of the crescent-shaped VNO, Although VNO240 was detected in neuronal perikarya at this age, it was not observed in the axons in the accessory olfactory bulb until postnatal day 3.5, This delayed appearance in the accessory olfactory bulb suggests that VNO240 is involved in the functional maturation of VNO neurones rather than in axon growth and targeting to the bulb, During the first 2 postnatal weeks, the population of neurones expressing VNO240 spread peripherally, and by adulthood all primary sensory neurones in the VNO appeared to be expressing this molecule. Similar patterns of expression were also observed for NOC-1, a previously characterized glycoform of the neural cell adhesion molecule NCAM, To date, differential expression of VNO-specific molecules has only been reported along the rostrocaudal axis or at different apical-basal levels in the neuroepithelium. This is the first demonstration of a centroperipheral wave of expression of molecules in the VNO, These results indicate that mechanisms controlling the molecular differentiation of VNO neurones must involve spatial cues organised, not only about orthogonal axes, but also about a centroperipheral axis, Moreover, expression about this centroperipheral axis also involves a temporal component because the subpopulation of neurones expressing VNO240 and NOC-1 increases during postnatal maturation. (C) 2001 John Wiley & Sons, Inc.

The ratio of messenger RNA levels of receptor activator of nuclear factor kappa B ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis

skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor KB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA, (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)Glc-Nac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

Surface diffusion of adsorbed molecules in porous media: Monolayer, multilayer, and capillary condensation regimes

This review provides an overview of surface diffusion and capillary condensate flow in porous media. Emphasis has been placed on the distinction between purely surface diffusion, multilayer surface diffusion, and, capillary condensate flow.

Olfaction in the Queensland fruit fly, Bactrocera tryoni. I: Identification of olfactory receptor neuron types responding to environmental odors

The electroantennogram method was used to investigate the number of distinct olfactory receptor neuron types responding to a range of behaviorally active volatile chemicals in gravid Queensland fruit flies, Bactrocera tryoni. Three receptor neuron types were identified. One type responds to methyl butyrate, 2-butanone, farnesene, and carbon dioxide; a second to ethanol; and a third to n-butyric acid and ammonia. The receptor neuron type responding to methyl butyrate, 2-butanone, farnesene, and carbon dioxide consists of three subtypes. The presence of a limited number of receptor neuron types responding to a diverse set of chemicals and the reception of carbon dioxide by a receptor neuron type that responds to other odorants are novel aspects of the peripheral olfactory discrimination process.

Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor

The cyclic C5a receptor antagonist, phenylalanine [L-ornithine-proline-D-cyclohexylalanine-tryptophan-arginine] (F-[OPchaWR]), has similar to 1000-fold less affinity for the C5a receptor (C5aR) on murine polymorphonuclear leukocytes than on human. Analysis of C5aR from different species shows that a possible cause of this difference is the variation in the sequence of the first extracellular loop of the receptor. The mouse receptor contains Y at a position analogous to P-103 in the human receptor, and D at G(105). To test this hypothesis, we expressed human C5aR mutants ((PY)-Y-103, G(105)D and the double mutant, (PY)-Y-103/G(105)D) in RBL-2H3 cells and investigated the effects of these mutations on binding affinity and receptor activation. All three mutant receptors had a higher affinity for human C5a than the wild-type receptor, but showed no significant difference in the ability of F-[OPchaWR] to inhibit human C5a binding. However, all of the mutant receptors had substantially lower affinities for the weak agonist, C5a des Arg(74) (C5adR(74)), and two altered receptors (G(105)D and (PY)-Y-103/G(105)D) had much lower affinities for the C-terminal C5a agonist peptide analogue, L-tyrosine-serine-phenylalanine-lysine-proline-methionine-proline-leucine-D-alanine-arginine (YSFKPMPLaR). Although it is unlikely that differences at these residues are responsible for variations in the potency of F-[OPchaWR] across species, residues in the first extracellular loop are clearly involved in the recognition of both C5a and C5a agonists. The complex effects of mutating these residues on the affinity and response to C5a, C5adR(74), and the peptide analogues provide evidence of different binding modes for these ligands on the C5aR. (C) 2001 Elsevier Science Inc. All rights reserved.