CB1 cannabinoid receptor-phosphorylated fourth intracellular loop structure-function relationships.

A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three Serine residues (Ser402, Ser411 and Ser415). Here we report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism, G protein activation and cAMP production. Circular dichroism studies indicated that phosphorylation at various Ser residues induced helical structure in different environments. NMR data indicates that helical content varies in the order of IL4pSer411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells were correlated with helicity changes. Treatment of cells with bradykinin, which activates PKC, augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor, suggesting that phosphorylation of serine might be a physiologically relevant modification in vivo. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can increase efficacy of G protein signaling.

The association between the PTPN22 C1858T polymorphism and systemic lupus erythematosus: a meta-analysis update.

The aim of this study was to determine whether the functional protein tyrosine phosphatase nonreceptor 22 (PTPN22) C1858T polymorphism (rs2476601) confers susceptibility to systemic lupus erythematosus (SLE) in ethnically different populations. A meta-analysis was conducted on the PTPN22 C1858T polymorphism across 11 comparative studies. Meta-analysis showed an association between the PTPN22 1858T allele and SLE in all study subjects (odds ratio (OR) 1.560, 95% confidence interval (CI) 1.336, 1.822, p = 2.0 × 10(-8)). Analysis after stratification by ethnicity indicated that the PTPN22 1858T allele was significantly associated with SLE in Europeans and Hispanics (OR 1.490, 95% CI 1.280, 1.735, p = 2.0 ×10(-8); OR 2.355, 95% CI 1.644, 3.373, p = 2.9 × 10(-6)). The meta-analysis showed that the C/T + T/T genotype was associated with susceptibility to SLE in all study subjects, Europeans, and Hispanics populations, and an association between the T/T genotype with SLE in Europeans. African Americans had a much lower prevalence of the T allele (2.2%) than any other population studied, and Europeans had the highest frequency (9.5%). In conclusion, this meta-analysis confirms that the PTPN22 C1858T polymorphism is associated with SLE susceptibility in different ethnic groups, and that its prevalence is ethnicity dependent.

Binding mode prediction of conformationally restricted anandamide analogs within the CB1 receptor.

BACKGROUND: CB1 cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and 2-arachidonoylglycerol. Because these arachidonic acid metabolites possess a 20-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated. RESULTS: A CB1 receptor model was constructed to include the extracellular loops, particularly extracellular loop 2 which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55244. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55244. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB1 receptor. CONCLUSION: Analyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB1 receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues.

Acylation of N-p-Toluenesulfonylpyrrole Under Friedel-Crafts Conditions. Evidence for Organoaluminum Intermediates.

The Friedel-Crafts acylation of N-p-toluenesulfonylpyrrole under Friedel-Crafts conditions has been reinvestigated. Evidence is presented in support of the hypothesis that when AlCl(3) is used as the Lewis acid, acylation proceeds via reaction of an organoaluminum intermediate with the acyl halide. This leads to the production of the 3-acyl derivative as the major product. With weaker Lewis acids (EtAlCl(2), Et(2)AlCl) or less than one equivalent of AlCl(3) the relative amount of 2-acyl product is increased. A mechanistic rationalization is presented to explain these data.

Modulation of angiotensin II and norepinephrine-induced plasminogen activator inhibitor-1 expression by AT1a receptor deficiency.

Angiotensin (Ang) II stimulates plasminogen activator inhibitor-1 (PAI-1) expression in many cell types by mechanisms that are cell-type specific. We measured effects of Ang II or norepinephrine on PAI-1 expression in wild type (WT) and Ang type-1a receptor knockout mice (AT(1a)-/-) in the presence or absence of the non-specific AT(1) antagonist losartan. Ang II and norepinephrine increased systolic blood pressure equally, whereas losartan decreased the pressor response of the former but not the latter in WT mice. In AT(1a)-/- mice, baseline systolic blood pressure was lower with no effect of Ang II, norepinephrine, or losartan. Ang II stimulated PAI-1 expression in the heart, aorta, and kidney and markedly in the liver of WT mice. In AT(1a)-/- mice, Ang II-stimulated PAI-1 was significantly attenuated compared with the WT in the heart and aorta but significantly enhanced in the kidney. Losartan decreased the induction in the aorta and liver of WT, and in the kidney and liver of AT(1a)-/- mice. Norepinephrine increased PAI-1 expression in WT heart and aorta, and in AT(1a)-/- heart, kidney, and liver with no effect of losartan. Renal PAI-1 expression correlated with AT(1b) receptor mRNA. We conclude that Ang II stimulates PAI-1 expression in part through the AT(1b) receptor in the kidney and liver. Further, norepinephrine induces PAI-1 expression in vivo with AT(1a) receptor deficiency modulating the effect.

The expanding role of epigenetics in the development, diagnosis and treatment of prostate cancer and benign prostatic hyperplasia.

PURPOSE: Prostate cancer research has focused significant attention on the mutation, deletion or amplification of the DNA base sequence that encodes critical growth or suppressor genes. However, these changes have left significant gaps in our understanding of the development and progression of disease. It has become clear that epigenetic changes or modifications that influence phenotype without altering the genotype present a new and entirely different mechanism for gene regulation. Several interrelated epigenetic modifications that are altered in abnormal growth states are DNA methylation changes, histone modifications and genomic imprinting. We discuss the status of epigenetic alterations in prostate cancer and benign prostatic hyperplasia progression. In addition, the rationale and status of ongoing clinical trials altering epigenetic processes in urological diseases are reviewed. MATERIALS AND METHODS: An online search of current and past peer reviewed literature on DNA methylation, histone acetylation and methylation, imprinting and epigenetics in prostate cancer and benign prostatic hyperplasia was performed. Relevant articles and reviews were examined and a synopsis of reproducible data was generated with the goal of informing the practicing urologist of these advances and their implications. RESULTS: Only 20 years ago the first study was published demonstrating global changes in DNA methylation patterns in tumors. Accumulating data have now identified specific genes that are commonly hypermethylated and inactivated during prostate cancer progression, including GSTpi, APC, MDR1, GPX3 and 14-3-3sigma. Altered histone modifications, including acetylation and methylation, were also recently described that may modify gene function, including androgen receptor function. These epigenetic changes are now being used to assist in prostate cancer diagnosis and cancer outcome prediction. Epigenetic changes appear to have a role in benign prostatic hyperplasia development as well as in the susceptibility of the prostate to developing cancer. Treatments involving 5-aza-deoxycytosine and other, more selective DNA methyltransferase inhibitors remove methyl residues from silenced genes, generating re-expression, and are currently being used in therapeutic trials. Histone deacetylase inhibitors have shown promise, not only by directly reactivating silenced genes, but also as regulators of apoptosis and sensitizers to radiation therapy. CONCLUSIONS: Evolving data support a significant role for epigenetic processes in the development of prostate cancer and benign prostatic hyperplasia. Epigenetic changes can predict tumor behavior and often distinguish between genetically identical tumors. Targeted drugs that alter epigenetic modifications hold promise as a tool for curing and preventing these diseases.

1-Alkyl-2-aryl-4-(1-naphthoyl)pyrroles: new high affinity ligands for the cannabinoid CB1 and CB2 receptors.

Two series of 1-alkyl-2-aryl-4-(1-naphthoyl)pyrroles were synthesized and their affinities for the cannabinoid CB(1) and CB(2) receptors were determined. In the 2-phenyl series (5) the N-alkyl group was varied from n-propyl to n-heptyl. A second series of 23 1-pentyl-2-aryl-4-(1-naphthoyl)-pyrroles (6) was also prepared. Several compounds in both series have CB(1) receptor affinities in the 6-30nM range. The high affinities of these pyrrole derivatives relative to JWH-030 (1, R=C(5)H(11)) support the hypothesis that these pyrroles interact with the CB(1) receptor primarily by aromatic stacking.

Recent developments in cannabinoid ligands.

Over the past 40 years, much research has been carried out directed toward the characterization of the cannabinergic system. With the identification of two G-protein coupled receptors and the endogenous ligand, anandamide, pharmacological targets have expanded to encompass hydrolase and transport proteins as well as novel classes of cannabinoid ligands. Those ligands that demonstrate high affinity for the receptors and good biological efficacy are tied together through lipophilic regions repeatedly demonstrated necessary for activity. This review presents recent developments in the structure-activity relationships of several classes of cannabinoid ligands.

Long-chain acyl-CoA dehydrogenase is a key enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids.

The first reaction of mitochondrial beta-oxidation, which is catalyzed by acyl-CoA dehydrogenases, was studied with unsaturated fatty acids that have a double bond either at the 4,5 or 5,6 position. The CoA thioesters of docosahexaenoic acid, arachidonic acid, 4,7,10-cis-hexadecatrienoic acid, 5-cis-tetradecenoic acid, and 4-cis-decenoic acid were effectively dehydrogenated by both rat and human long-chain acyl-CoA dehydrogenases (LCAD), whereas they were poor substrates of very long-chain acyl-CoA dehydrogenases (VLCAD). VLCAD, however, was active with CoA derivatives of long-chain saturated fatty acids or unsaturated fatty acids that have double bonds further removed from the thioester function. Although bovine LCAD effectively dehydrogenated 5-cis-tetradecenoyl-CoA (14:1) and 4,7,10-cis-hexadecatrienoyl-CoA, it was nearly inactive toward the other unsaturated substrates. The catalytic efficiency of rat VLCAD with 14:1 as substrate was only 4% of the efficiency determined with tetradecanoyl-CoA, whereas LCAD acted equally well on both substrates. The conclusion of this study is that LCAD serves an important, if not essential function in the beta-oxidation of unsaturated fatty acids.

Comparison between electroglottography and electromagnetic glottography.

Newly developed glottographic sensors, utilizing high-frequency propagating electromagnetic waves, were compared to a well-established electroglottographic device. The comparison was made on four male subjects under different phonation conditions, including three levels of vocal fold adduction (normal, breathy, and pressed), three different registers (falsetto, chest, and fry), and two different pitches. Agreement between the sensors was always found for the glottal closure event, but for the general wave shape the agreement was better for falsetto and breathy voice than for pressed voice and vocal fry. Differences are attributed to the field patterns of the devices. Whereas the electroglottographic device can operate only in a conduction mode, the electromagnetic device can operate in either the forward scattering (diffraction) mode or in the backward scattering (reflection) mode. Results of our tests favor the diffraction mode because a more favorable angle imposed on receiving the scattered (reflected) signal did not improve the signal strength. Several observations are made on the uses of the electromagnetic sensors for operation without skin contact and possibly in an array configuration for improved spatial resolution within the glottis.

Inhibition of nuclear factor-kappaB-mediated adhesion molecule expression in human endothelial cells.

The transcriptional regulatory protein nuclear factor-kappaB (NF-kappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses, including the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1). NF-kappaB is found in the cytoplasm complexed with its inhibitory protein IkappaB. On activation, IkappaB is phosphorylated and degraded, thereby freeing NF-kappaB for translocation to the nucleus. We have generated populations of endothelial cells expressing wild-type and a proteolysis-resistant mutation of IkappaB that is lacking the 36 N-terminal amino acids (IkappaBdeltaN) in order to examine the effects of expression of the mutated IkappaB on tumor necrosis factor-alpha (TNF-alpha)-induced E-selectin and ICAM-1 expression. Wild-type and IkappaBdeltaN were introduced into primary endothelial cells using retrovirus infection followed by selection with G418. The IkappaBdeltaN protein remained at untreated control levels in endothelial cells treated with TNF-alpha and also remained complexed with the NF-kappaB family member p65. Furthermore, TNF-alpha-induced NF-kappaB DNA binding activity was inhibited in the population of endothelial cells expressing IkappaBdeltaN. That population of cells was also refractory to upregulation of E-selectin and ICAM-1 after treatment with TNF-alpha. The use of a truncated IkappaBalpha protein to prevent NF-kappaB-mediated gene expression provides a novel and specific approach for investigating the role of NF-kappaB in processes associated with adhesion molecule expression during inflammation.

Speech articulator measurements using low power EM-wave sensors.

Very low power electromagnetic (EM) wave sensors are being used to measure speech articulator motions as speech is produced. Glottal tissue oscillations, jaw, tongue, soft palate, and other organs have been measured. Previously, microwave imaging (e.g., using radar sensors) appears not to have been considered for such monitoring. Glottal tissue movements detected by radar sensors correlate well with those obtained by established laboratory techniques, and have used to estimate a voiced excitation function for speech processing applications. The noninvasive access, coupled with the small size, low power, and high resolution of these new sensors, permit promising research and development applications in speech production, communication disorders, speech recognition and related topics.

Immunihistochemical detection of Bcl-2 in AIDS-associated and classical Kaposi's sarcoma.

Kaposi's Sarcoma (KS) is an angioproliferative disease that is characterized by proliferation of spindle-shaped cells predominantly of vascular endothelial cell origin, neoangiogenesis, inflammatory cell infiltration, and edema. Although the lesions of classical KS and AIDS-associated KS (AIDS-KS) share common histological features, AIDS-KS occurs at a markedly higher frequency with a more aggressive clinical course. Immunohistochemical analyses of 26 evolutionarily staged AIDS-KS lesions derived from HIV-infected patients demonstrate significant cytoplasmic levels of Bcl-2, a protooncogene known to prolong cellular viability and to antagonize apoptosis. Bcl-2 expression increases as the pathological stage of KS advances. Immunohistochemical analyses of classical KS lesions demonstrate prevalent expression of Bcl-2 as well, indicating that upregulation of Bcl-2 may be important in the pathogenesis of both classical and AIDS-associated KS. Coexpression of Bcl-2 and factor VIII-related antigen in spindle-shaped cells present within KS lesions suggests that Bcl-2 is upregulated within the vascular endothelial spindle-shaped cells of KS. The consequences of upregulated Bcl-2 expression within KS lesions may be prolonged spindle cell viability which, when coupled with dysregulated cellular proliferation due in part to synergistic activities of inflammatory and angiogenic cytokines and HIV-1 Tat protein, may result in the maintenance, growth, and progression of KS.