Binding mechanisms for Thermobifida fusca Cel5A,Cel6B,and Cel48A cellulose-binding modules on bacterial microcrystalline cellulose

The family II cellulose-binding modules (CBM) from Thermobifida fusca Cel5A and Cel48A were cloned in the Escherichia coli/Streptomyces shuttle vector pD730, and the plasmids were transformed into Streptomyces lividans TKM31. CBM(Cel5A), and CBM(Cel48A), CBM(Cel6B) were expressed and purified from S. lividans. The molecular masses were determined by mass spectrometry, and the values were 10595 +/- 2, 10915 +/- 2, and 11291 +/- 2 Da for CBM(Cel5A), CBM(Cel6B), and CBM(Cel48A), respectively. Three different binding models (Langmuir, Interstice Penetration, and Interstice Saturation) were tested to describe the binding isotherms of these CBMs on bacterial microcrystalline cellulose (BMCC). The experimental binding isotherms of T. fusca family II CBMs on BMCC are best modeled by the Interstice Saturation model, which includes binding to the constrained interstice surface of BMCC as well as traditional Langmuir binding on the freely accessible surface. The Interstice Saturation model consists of three different steps (Langmuir binding, interstice binding, and interstice saturation). Full reversibility only occurred in the Langmuir region. The irreversibility in the interstice binding and saturation regions probably was caused by interstice entrapment. Temperature shift experiments in different binding regions support the interstice entrapment assumption. There was no systematic difference in binding between the two types of exocellulase CBMs--one that hydrolyzes cellulose from the nonreducing (CBM(Cel6B)) end and one that hydrolyzes cellulose from the reducing end (CBM(Cel48A)).     

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Synthesis, structure–affinity relationships, and modeling of AMDA analogs at 5-HT2A and H1 receptors: Structural factors contributing to selectivity

Histamine H₁ and serotonin 5-HT_2A receptors present in the CNS have been implicated in various neuropsychiatric disorders. 9-Aminomethyl-9,10-dihydroanthracene (AMDA), a conformationally constrained diarylalkyl amine derivative, has affinity for both of these receptors. A structure–affinity relationship (SAFIR) study was carried out studying the effects of N-methylation, varying the linker chain length and constraint of the aromatic rings on the binding affinities of the compounds with the 5-HT_2A and H₁ receptors. Homology modeling of the 5-HT_2A and H₁ receptors suggests that AMDA and its analogs, the parent of which is a 5-HT_2A antagonist, can bind in a fashion analogous to that of classical H₁ antagonists whose ring systems are oriented toward the fifth and sixth transmembrane helices. The modeled orientation of the ligands are consistent with the reported site-directed mutagenesis data for 5-HT_2A and H₁ receptors and provide a potential explanation for the selectivity of ligands acting at both receptors.     

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT_2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT_2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.     

Binding and reversibility of Thermobifida fusca Cel5A,Cel6B,and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose

The binding and reversibility of Thermobifida fusca intact Cel5A, Cel5B, and Cel48A and their corresponding catalytic domains (CDs) to bacterial microcrystalline cellulose (BMCC) were studied at 5 degrees C. The binding of the intact cellulases and of corresponding CDs to BMCC was irreversible in all regions: Langmuir binding (region I), interstice penetration (region II), and interstice saturation (region III). The three cellulose binding domains (CBMs) bind reversibly in "region I" although their respective CDs do not. The irreversible binding of these enzymes in the Langmuir region does not satisfy the Langmuir assumption; however, the overall fit of the Interstice Saturation model, which includes binding in MBCC interstices as well as on the freely accessible surface (Jung et al., 2002a) is good. The main limitation of the model is that it does not explicitly address a mechanism for forming the enzyme-substrate complex within the active site of the CDs.     

Identification of Amino Acid Residues Responsible for the Selectivity of Tadalafil Binding to Two Closely Related Phosphodiesterases,PDE5 and PDE6

The 11 families of the Class I cyclic nucleotide phosphodiesterases (PDEs) are critical for regulation of cyclic nucleotide signaling. PDE5 (important in regulating vascular smooth muscle contraction) and PDE6 (responsible for regulating visual transduction in vertebrate photoreceptors) are structurally similar but have several functional differences whose structural basis is poorly understood. Using evolutionary trace analysis and structural homology modeling in conjunction with site-directed mutagenesis, we have tested the hypothesis that class-specific differences between PDE5 and PDE6 account for the biochemical and pharmacological differences in the two enzyme families. Replacing human PDE5 residues in the M-loop region of the binding site for the PDE5-selective inhibitor tadalafil (Cialis®) with the corresponding class-specific cone PDE6 residues (P773E, I778V, E780L, F787W, E796V, D803P, L804M, N806D, I813L, S815K) reduces tadalafil binding affinity to levels characteristic of PDE6. These mutations fail to alter vardenafil (Levitra®) affinity for the active site. Class-specific differences in PDE5 versus cone PDE6 that contribute to the accelerated catalytic efficiency of PDE6 were identified but required heterologous expression of full-length PDE5 constructs. Introduction of PDE6 residues into the background of the PDE5 protein sequence often led to loss of catalytic activity and reduced protein solubility, supporting the idea that multiple structural elements of PDE6 are highly susceptible to misfolding during heterologous expression. This work validates the use of PDE5 as a template to identify functional differences between PDE5 and PDE6 that will accelerate efforts to develop the next generation of PDE5-selective inhibitors with fewer adverse side effects resulting from PDE6 inhibition.     

Synthesis, crystal structure, spectral properties and catalytic activity of hexakis(imidazole)manganese(II) bisindole-2-carboxylato bisneocuproine bisdimethyl sulfoxide solvates {[Mn(Im)6] · 2(2-IC) · 2(NC) · 2(DMSO)}

Synthesis, X-ray crystal structure and IR spectrum of {[Mn^II(Im)₆] · 2(2-IC) · 2(NC) · 2(DMSO)} (Im = imidazole, 2-HIC = indole-2-carboxylic acid, NC = 2,9-dimethyl-1,10-phenanthroline, DMSO = dimethyl sulfoxide) are reported. The manganese(II) ion has octahedral geometry with a MnN₆ core. The crystal structure is completed by two NC, two 2-IC⁻ and two DMSO solvate molecules. The individual cations are linked into chains running parallel to the a axis by four intermolecular hydrogen bonding involving two 2-IC⁻solvate. Moreover, these chains are connected by π-π stacking interactions which occur between neocuproine molecules related through inversion center. In IR spectroscopy, the compound spectrum is roughly similar to the imidazole one: (i) above 1800 cm⁻¹, the bands are broad, but when focussing on some of them a doublet structure can be found; (ii) below 1800 cm⁻¹, the bands are sharp and it is then possible to point out the modification of S-O band when this later is involved in bifurcated hydrogen bonding to a second solvate 2-IC⁻. The compound catalyses the disproportionation of H₂O₂; moreover an additional quantity of imidazole increases the reaction rate.