Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

Cannabinoid ligand-receptor signaling in the mouse uterus.

Using RNA (Northern) blot hybridization and reverse transcription-PCR, we demonstrate that the brain-type cannabinoid receptor (CB1-R) mRNA, but not the spleen-type cannabinoid receptor (CB2-R) mRNA, is expressed in the mouse uterus and that this organ has the capacity to synthesize the putative endogenous cannabinoid ligand, anandamide (arachidonylethanolamide). The psychoactive cannabinoid component of marijuana--delta 9-tetrahydrocannabinol (THC)--or anandamide, but not the inactive and nonpsychoactive cannabidiol (CBD), inhibited forskolin-stimulated cyclic AMP formation in the mouse uterus, which was prevented by pertussis toxin pretreatment. These results suggest that uterine CB1-R is coupled to inhibitory guanine nucleotide-binding protein and is biologically active. Autoradiographic studies identified ligand binding sites ([3H]anandamide) in the uterine epithelium and stromal cells, suggesting that these cells are perhaps the targets for cannabinoid action. Scatchard analysis of the binding of [3H]WIN 55212-2, another cannabinoid receptor ligand, showed a single class of high-affinity binding sites in the endometrium with an apparent Kd of 2.4 nM and Bmax of 5.4 x 10(9) molecules per mg of protein. The gene encoding lactoferrin is an estrogen-responsive gene in the mouse uterus that was rapidly and transiently up-regulated by THC, but not by CBD, in ovariectomized mice in the absence of ovarian steroids. This effect, unlike that of 17 beta-estradiol (E2), was not influenced by a pure antiestrogen, ICI 182780, suggesting that the THC-induced uterine lactoferrin gene expression does not involve estrogen receptors. We propose that the uterus is a new target for cannabinoid ligand-receptor signaling.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

Differential expression of N-methyl-D-aspartate receptor NR2 isoforms in Alzheimer's disease

We have previously shown that the expression of NMDA receptor NR1 subunit mRNA splice variants in Alzheimer's disease (AD) brain varies according to regional susceptibility to pathological damage. Here we investigated the expression of the modulatory NR2 subunits of the NMDA receptor using quantitative RT-PCR to assay all NR2 isoforms. Significantly lower expression of NR2A and NR2B transcripts was found in susceptible regions of AD brain, whereas expression of NR2C and NR2D transcripts did not differ from that in controls. Western blot analysis confirmed a lower expression of the NR2A and NR2B isoforms at the protein level. The results suggest that NR2 subunit composition may modulate NMDA receptor-mediated excitotoxicity. NMDA receptor dysfunction might give rise to the regionally selective pattern of neuronal loss that is characteristic of AD.

Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol

Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with mbeta-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-alpha activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKCalpha, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane.

Potent cyclic antagonists of the complement C5a receptor on human polymorphonulcear leukocytes. Relationships between structures and activity

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC50, 20 nM(-1) muM). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinity-determining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G protein-coupled receptor.

Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters

Cone snails have evolved a vast array of peptide toxins for prey capture and defence. These peptides are directed against a wide variety of pharmacological targets, making them an invaluable source of ligands for studying the properties of these targets in normal and diseased states. A number of these peptides have shown efficacy in vivo, including inhibitors of calcium channels, the norepinephrine transporter, nicotinic acetylcholine receptors, NMDA receptors and neurotensin receptors, with several having undergone pre-clinical or clinical development for the treatment of pain.

Increased potency of a novel complement factor 5a receptor antagonist in a rat model of inflammatory bowel disease

We have previously shown that complement factor 5a(C5a) plays a role in the pathogenesis of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats by using the selective, orally active C5a antagonist AcF-[OP(D-Cha) WR]. This study tested the efficacy and potency of a new C5a antagonist, hydrocinnamate (HC)-[OP(D-Cha) WR], which has limited intestinal lumenal metabolism, in this model of colitis. Analogs of AcF-[OP(D-Cha) WR] were examined for their susceptibility to alimentary metabolism in the rat using intestinal mucosal washings. One metabolically stable analog, HC-[OP(D-Cha)WR], was then evaluated pharmacokinetically and investigated at a range of doses (0.03 - 10 mg/kg/ day p.o.) in the 8-day rat TNBS- colitis model, against the comparator drug AcF-[OP(D-Cha) WR]. Using various amino acid substitutions, it was determined that the AcF moiety of AcF-[OP(D-Cha) WR] was responsible for the metabolic instability of the compound in intestinal mucosal washings. The analog HC-[OP( D-Cha) WR], equiactive in vitro to AcF-[OP(D-Cha) WR], was resistant to intestinal metabolism, but it displayed similar oral bioavailability to AcF-[OP(D-Cha) WR]. However, in the rat TNBS- colitis model, HC-[OP(D-Cha) WR] was effective at reducing mortality, colon edema, colon macroscopic scores, and increasing food consumption and body weights, at 10- to 30- fold lower oral doses than AcF-[OP( D-Cha) WR]. These studies suggest that resistance to intestinal metabolism by HC-[OP(D-Cha) WR] may result in increased local concentrations of the drug in the colon, thus affording efficacy with markedly lower oral doses than AcF-[OP(D-Cha) WR] against TNBS-colitis. This large increase in potency and high efficacy of this compound makes it a potential candidate for clinical development against intestinal diseases such as inflammatory bowel disease.

Developmental changes in expression of GABA(A) receptor-channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was similar to 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

Nur77 regulates lipolysis in skeletal muscle cells - Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway

Skeletal muscle is a major mass peripheral tissue that accounts for similar to 40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces beta-adrenergic receptor (beta-AR)- mediated adaptive thermogenesis. beta-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30 - 60 min of beta-AR agonist ( isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (> 100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP-activated protein kinase gamma 3, UCP3, CD36,adiponectin receptor 2, GLUT4, and caveolin-3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross-talk between the nuclear hormone receptor and beta-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity.

Rev-erb beta regulates the expression of genes involved in lipid absorption in skeletal muscle cells - Evidence for cross-talk between orphan nuclear receptors and myokines

Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Rev-erbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for > 30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (> 15-fold) in mRNA expression of interleukin-6, an exercise-induced myokine that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (> 20- fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.

Epidermal penetration of a therapeutic peptide by lipid conjugation; Stereo-selective peptide availability of a topical diastereomeric lipopeptide

In inflammatory disorders (e.g. psoriasis), local concentrations of human neutrophil elastase (HNE), also known as polymorphonuclear leukocyte elastase (HLE), possibly overwhelm its natural inhibitors leading to extracellular matrix degradation. Elevated levels of HNE have been reported in a variety of inflammatory disorders, including psoriasis. Peptidic HNE inhibitors have a common hydrophobic sequence (Ala-Ala-Pro-Val). This peptide sequence inhibits HNE competitively; however the stratum corneum presents an effective barrier to the delivery of this tetrapeptide across the skin. The current work investigates the delivery of the modified peptide whereby the tetrapeptide was lipidated to enhance its ability to penetrate the stratum The tetrapeptide Was Coupled to a racaemic mixture of a short chain lipoamino acid (LAA) resulting in two diastereomers of the lipoamino acid-modified tetrapeptide. The penetration of the lipopeptide mixture was assessed across human epidermis in vitro. The percentage of applied dose penetrating to the receptor over 8 h following administration was 2.53% for the D-LAA conjugate and 1.47% for the L-diastereomer, compared to 0% for the peptide. The D-diastereomer appears to be relatively stable but the L-diastereomer appears to degrade releasing possibly the tetrapeptide and peptide fragment(s). Therefore the results clearly indicate that coupling the tetrapeptide to a short chain LAA enhances its delivery across human epidermis.

Chronic smoking and alcoholism change expression of selective genes in the human prefrontal cortex

Background: Alcoholism is commonly associated with chronic smoking. A number of gene expression profiles of regions within the human mesocorticolimbic system have identified potential alcohol-sensitive genes; however, the influence of smoking on these changes was not taken into account. This study addressed the impact of alcohol and smoking on the expression of 4 genes, previously identified as alcoholism-sensitive. in the human prefrontal cortex (PFC). Methods: mRNA expression of apolipoprotein D, tissue inhibitor of the metalloproteinase 3, high-affinity glial glutamate transporter and midkine, was measured in the PFC of alcoholic Subjects and controls with and without smoking comorbidity using real-time polymerase chain reaction. Results: The results show that alcohol affects transcription of some of these genes. Additionally, smoking has a marked influence on gene expression. Conclusion: This study emphasizes the need for careful case selection in future gene expression studies to delineate the adaptive molecular process associated with smoking and alcohol.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.