Stirrer tank: an appropriate technology to immobilize the CB.Hep-1 monoclonal antibody for immunoaffinity purification

The CB.Hep-1 monoclonal antibody was coupled to CNBr-activated Sepharose CL 4B at three different immobilization scales for purification of recombinant hepatitis B surface antigen. Standard laboratory apparatus to obtain immunosorbents of 1 l (scale I) and 3 l (scale II) as well as a stirrer tank to prepare 6 l immunosorbents (scale III) were used. The binding capacity at scale III was 2- and 1.5-fold higher with respect to the scales II and I, while a reduction in the ligand leakage of 5- and 2-folds was observed. Immunosorbents from scale II showed a significantly reduced adsorption, and an increased ligand leakage. Differences in the coupling efficiency were not observed. Antigen purity eluted from the immunosorbents was always above 85%.     

CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor

The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg ml^-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml^-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics of the mAb harvested from the bioreactor during the 43 days of cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis and testing of novel classical cannabinoids: exploring the side chain ligand binding pocket of the CB1 and CB2 receptors

A series of C3 cyclic side-chain analogues of classical cannabinoids were synthesized to probe the ligand binding pocket of the CB1 and CB2 receptors. The analogues were evaluated for CB1 and CB2 receptor binding affinities relative to delta(8)-THC. The C3 side-chain geometries of the analogues were studied using high field NMR spectroscopy and quantum mechanical calculations. The results of these studies provide insights into the geometry of the ligand binding pocket of the CB1 and CB2 receptors.     

Exploring the first Rimonabant analog-opioid peptide hybrid compound,as bivalent ligand for CB1 and opioid receptors

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH₂. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.     

MBC94, a conjugable ligand for cannabinoid CB 2 receptor imaging

Cannabinoid CB 2 receptor is a particularly attractive target for noninvasive imaging of neuroinflammation and monitoring of therapeutic efficacy. Its expression is low to undetectable in healthy brain and induced in resident microglial cells (the macrophage of the brain) after cerebral ischemia, injury, and in neuroinflammatory disease. Additionally, immune cells migrating across the blood-brain barrier typically express CB 2 receptors, which adds to the expression pool of this target and provides a reliable indicator of inflammation in the brain. Here, we synthesized a novel conjugable CB 2 receptor ligand, mbc94, which has a terminal amino group that allows for facile conjugation to imaging moieties. A near-infrared (NIR) dye labeled mbc94, NIRmbc94, was developed for CB 2 targeted imaging. Preliminary evidence, including in vitro fluorescence imaging and a competition study, showed that NIRmbc94 specifically labeled CB 2-expressing cells.     

Comparative molecular dynamics simulations of the potent synthetic classical cannabinoid ligand AMG3 in solution and at binding site of the CB1 and CB2 receptors

The C-1'-dithiolane Delta(8)-tetrahydrocannabinol (Delta(8)-THC) amphiphilic analogue (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethylhydroxy-6H-dibenzo[b,d]pyranyl)-2-hexyl-1,3-dithiolane (AMG3) is considered as one of the most potent synthetic analgesic cannabinoid (CB) ligands. Its structure is characterized by rigid tricyclic and flexible alkyl chain segments. Its conformational properties have not been fully explored. Structure-activity relationship (SAR) studies on classical CBs showed that the alkyl side chain is the most critical structural part for the receptor activation. However, reported low energy conformers of classical CB analogues vary mainly in the conformation of their alkyl side chain segment. Therefore, comparative molecular dynamics (MD) simulations of low energy conformers of AMG3 were performed in order to investigate its structural and dynamical properties in two different systems. System-I includes ligand and amphoteric solvent DMSO, simulating the biological environment and system-II includes ligand at active site of the homology models of CB1 and CB2 receptors in the solvent. The trajectory analysis results are compared for the systems I and II. In system-I, the dihedral angle defined between aromatic ring and dithiolane ring of AMG3 shows more resistance to be transformed into another torsional angle and the dihedral angle adjacent to dithiolane ring belonging in the alkyl chain has flexibility to adopt gauche+/- and trans dihedral angles. The rest of the dihedral angles within the alkyl chain are all trans. These results point out that wrapped conformations are dynamically less favored in solution than linear conformations. Two possible plane angles defined between the rigid and flexible segments are found to be the most favored and adopting values of approximately 90 degrees and approximately 140 degrees. In system-II, these values are approximately 90 degrees and approximately 120 degrees. Conformers of AMG3 at the CB1 receptor favor to establish a cis conformation defined between aromatic and dithiolane ring and a trans conformation in the CB2 receptor. These different orientations of ligand inside the binding pocket of CB1 and CB2 receptors may explain its different binding affinity in the two receptors. The results of this study can be applied to other synthetic classical CB ligands to produce low energy conformations and can be of general use for the molecules possessing flexible alkyl chain(s). In addition, this study can be useful when restraint of the alkyl chain is sought for optimizing drug design.     

Hypersensitization of the Orexin 1 receptor by the CB1 receptor: evidence for cross-talk blocked by the specific CB1 antagonist,SR141716

In the present study, we observed evidence of cross-talk between the cannabinoid receptor CB1 and the orexin 1 receptor (OX1R) using a heterologous system. When the two receptors are co-expressed, we observed a major CB1-dependent enhancement of the orexin A potency to activate the mitogen-activated protein kinase pathway; dose-responses curves indicated a 100-fold increase in the potency of orexin-mediated mitogen-activated protein kinase activation. This effect required a functional CB1 receptor as evidenced by the blockade of the orexin response by the specific CB1 antagonist, N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716), but also by pertussis toxin, suggesting that this potentiation is Gi-mediated. In contrast to OX1R, the potency of direct activation of CB1 was not affected by co-expression with OX1R. In addition, electron microscopy experiments revealed that CB1 and OX1R are closely apposed at the plasma membrane level; they are close enough to form hetero-oligomers. Altogether, for the first time our data provide evidence that CB1 is able to potentiate an orexigenic receptor. Considering the antiobesity effect of SR141716, these results open new avenues to understand the mechanism by which the molecule may prevent weight gain through functional interaction between CB1 and other receptors involved in the control of appetite.     

Imidazopyridine CB2 agonists: optimization of CB2/CB1 selectivity and implications for in vivo analgesic efficacy

A new series of imidazopyridine CB2 agonists is described. Structural optimization improved CB2/CB1 selectivity in this series and conferred physical properties that facilitated high in vivo exposure, both centrally and peripherally. Administration of a highly selective CB2 agonist in a rat model of analgesia was ineffective despite substantial CNS exposure, while administration of a moderately selective CB2/CB1 agonist exhibited significant analgesic effects.     

Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.

Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (anandamide), methylated at the 2,1' and 2' positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1'-dimethyl analogues. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (4) exhibited the highest CB1 receptor affinity in this series with a K(i) of 7.42 nM, an at least 10-fold improvement on anandamide (K(i)=78.2 nM). The introduction of two methyl groups at the 2-position of anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2-gem-dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the (S)-2,2,2'-trimethyl analogue (12) had the highest affinity for CB1 almost equal to that of anandamide. In agreement with our previous anandamide structure-activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1'- and 2-methyl groups     

Production of a monoclonal antibody by ascites, hollow fiber system, and transgenic plants for vaccine production using CB.Hep-1 mAb as a study case

Monoclonal antibody (mAb) production methods (ascites, in vitro technologies, transgenic animals, and dicot or monocot transgenic plants; moss, algae) have been improved since they were first developed in 1975. In this study, we illustrate a summary of a study case in which mice, a hollow fiber system, and tobacco transgenic plants were assessed for the production of mAb for vaccine manufacturing and vaccine production.     

Use of immunosorbents for determining antibodies to histaglobulin]

A determination was made of antibodies to histaglobulin and gamma-globulin with the aid of immunosrobents in 178 sera of children suffering from allergic diseases, during the histaglobulin therapy. Results of investigations showed antibodies to histaglobulins to be absent in children untreated by this preparation. But they appeared in 57% of cases after the first course of histaglobulin treatment, and their incidence and their average level increased with an increase in the number of the courses of treatment carried out. gamma-Globulin antibodies were found at the initial condition in 42% of cases; this percentage rose to 68 after the first course of histoglobulin treatment. The authors believe that determination of histaglobulin antibodies during the treatment with this preparation could serve as an auxiliary immunological criterion of the efficacy of histaglobulin therapy.     

CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9–3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.     

Use of quantitative immunofluorescence for determining the adsorption capacity of magnetic immunosorbents

The adsorption capacity of microgranulated polyacrylamide magnetic immunosorbents has been studied by the method of quantitative immunofluorescence as applied to the causative agents of plague, cholera, and melioidosis. Similar regularity in the dynamics of antigen adsorption on the granules of magnetic immunoadsorbents has been established. This regularity consists in the direct relationship between this process and the concentration of infective agents interacting with magnetic sorbents.     

A comparison of some activated matrices for preparation of immunosorbents

The adsorption characteristics of monoclonal anti-(β-galactosidase) immobilised to a number of commercially available pre-activated matrices have been investigated in a series of small scale experiments. Binding characteristics were determined by batch isotherm techniques and estimates were obtained of the rate constants governing adsorption to the immobilised antibodies. The capacity of the different matrices for binding antibody and the specific activity of immunosorbents were measured.There was little effect of support matrix on the dissociation constant, K_d, for the interaction between β-galactosidase and immobilised anti-(β-galactosidase). However, the maximum amounts of antibody that could be immobilised, rates of adsorption and desorption of the enzyme to the immobilised antibody and the specific activity of immunosorbents were affected by the choice of support matrix. The importance of the relative sizes of the antigen and immobilised antibody and the influence of the nature of the support matrix on the properties of the resulting immunosorbent when used in large scale applications are discussed.     

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.     

苹果蠹蛾性信息素缓释剂的控害效果

2010年在甘肃、宁夏、黑龙江3地对苹果蠹蛾Cydia pomonella(L.)性信息素缓释剂(购自澳大利亚Bioglobal公司;性信息素含量为0.16g/根)对苹果蠹蛾的迷向防治效果进行了试验;观察了不同生态区设置不同缓释剂密度的防治效果和持效期。结果表明,在低虫口密度下,在不同生态区每公顷悬挂660、990和1320根性信息素迷向丝,均能取得较好的防治效果;在越冬代活动前处理,基本上可以控制整个生长期苹果蠹蛾对果实的为害。在新侵入的低密度果园,通过诱蛾监测、调查蛀果和树干绑诱虫带等验证方法,初步证明使用该性信息素缓释剂处理1年可以达到铲除的效果。依据本研究结果,结合防治成本,在苹果蠹蛾发生地区,采用660根/hm2的悬挂密度使用进口性信息素缓释剂处理果园可以达到良好的防治效果。     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

Heterogeneity in chemotaxis of neutrophils with different densities

We investigated N-formyl-Methionyl-Leucyl-Phenylalanine (fMLP)-induced chemotaxis of neutrophils with different densities. FMLP-induced (10(-8)M) chemotaxis of neutrophils with lower density were significantly reduced when compared to neutrophils with higher density (p less than 0.05). These findings imply a relationship between the neutrophil density and chemotaxis.