17ß-estradiol (E2) in membranes: Orientation and dynamic properties.

Non-genomic membrane effects of estrogens are of great interest because of the diverse biological activities they may elicit. To further our understanding of the molecular features of the interaction between estrogenic hormones and membrane bilayers, we have determined the preferred orientation, location, and dynamic properties of 17ß-estradiol (E2) in two different phospholipid membrane environments using (2)H-NMR and 2D (1)H-(13)C HSQC in conjunction with molecular dynamics simulations. Unequivocal spectral assignments to specific (2)H labels were made possible by synthesizing six selectively deuterated E2 molecules. The data allow us to conclude that the E2 molecule adopts a nearly "horizontal" orientation in the membrane bilayer with its long axis essentially perpendicular to the lipid acyl-chains. All four rings of the E2 molecule are located near the membrane interface, allowing both the E2 3-OH and the 17ß-OH groups to engage in hydrogen bonding and electrostatic interactions with polar phospholipid groups. The findings augment our knowledge of the molecular interactions between E2 and membrane bilayer and highlight the asymmetric nature of the dynamic motions of the rigid E2 molecule in a membrane environment.

The interaction of cannabinoid receptor agonists, CP55940 and WIN55212-2 with membranes using solid state 2H NMR.

Two key commonly used cannabinergic agonists, CP55940 and WIN55212-2, are investigated for their effects on the lipid membrane bilayer using (2)H solid state NMR, and the results are compared with our earlier work with delta-9-tetrahydrocannabinol (Δ(9)-THC). To study the effects of these ligands we used hydrated bilayers of dipalmitoylphosphatidylcholine (DPPC) deuterated at the 2' and 16' positions of both acyl chains with deuterium atoms serving as probes for the dynamic and phase changes at the membrane interface and at the bilayer center respectively. All three cannabinergic ligands lower the phospholipid membrane phase transition temperature, increase the lipid sn-2 chain order parameter at the membrane interface and decrease the order at the center of the bilayer. Our studies show that the cannabinoid ligands induce lateral phase separation in the lipid membrane at physiological temperatures. During the lipid membrane phase transition, the cooperative dynamic process whereby the C-(2)H segments at the interface and center of the bilayer spontaneously reach the fast exchange regime ((2)H NMR timescale) is distinctively modulated by the two cannabinoids. Specifically, CP55940 is slightly more efficient at inducing liquid crystalline-type (2)H NMR spectral features at the membrane interface compared to WIN55212-2. In contrast, WIN55212-2 has a far superior ability to induce liquid crystalline-type spectral features at the center of the bilayer, and it increases the order parameter of the sn-1 chain in addition to the sn-2 chain of the lipids. These observations suggest the cannabinoid ligands may influence lipid membrane domain formations and there may be contributions to their cannabinergic activities through lipid membrane microdomain related mechanisms. Our work demonstrates that experimental design strategies utilizing specifically deuterium labeled lipids yield more detailed insights concerning the properties of lipid bilayers.

[A study of environmental changes on new snail habitat though multi-temporal relative radiometric normalized TM images].

OBJECTIVE: To study the environmental changes of new snail habitual by using multi-temporal relative radiometric normalized TM images taken in Wuzhoutou of Nanchang, Jiangxi province. METHODS: TM images of Poyang Lake were collected on 3rd April 1998, 16th April 2000 and 5th May 2004. Taking images in 2004 as a reference, the TM images in 1998 and 2000 were radiometric normalized. The images were analyzed to trace the vegetation background and snail historical information. RESULTS: The vegetation coverage rate of Wuzhaoutou on April 3rd in 1998 was 4.76%, but the vegetation coverage rates of Wuzhoutou on April 16th in 2000 and May 5th in 2004 were above 80%; the NDVI values of Wuzhoutou on April 3rd in 1998 were around -0.4 to 0.2, but the NDVI values of Wuzhoutou on April 16th in 2000 and May 5th in 2004 were mainly around 0.2 to 0.6, which had statistical difference with that in 1998 (q = 162.285321, P < 0.01 and q = 161.669725, P < 0.01). The greenness of three images have statistical differences(q = 178.679245, P < 0.01; q = 130.320755, P < 0.01 and q = - 48.358491, P < 0.01). CONCLUSION: Combining with the snail survey, the TM images could be an effective measure for setting up models to forecast the areas where snails might live to control the Schistosomiasis epidemic.

[Application of global positioning system and geographic information system in surveillance of schistosomiasis].

OBJECTIVE: To use the snail survey data of Yugan, Jiangxi Province as an example to evaluate the effects and advantages of global positioning system integrated with digital maps and remote sensing data in order to manage and analyze the whole country's surveillance data of schistosomiasis by using geographic information system. METHODS: The data of geographic information and snail information of 20 marshlands in Yugan, Jiangxi Province were collected, and by the correction of remote sensing images based on the digital maps and index extraction, the snail current situation and trend were analyzed and expressed. RESULTS: A series of specific procedure images of these 20 marshlands was established and the effects of Yugan surveillance platform based on geographic information system were validated. CONCLUSION: The whole country's surveillance data of schistosomiasis might be successfully integrated with geographic information system by global positioning system and be managed and expressed, which supplies a successful platform in using the surveillance data effectively.

Hydrogen-bonded His93 as a sensitive probe for identifying inhibitors of the endocannabinoid transport protein FABP7.

The human brain FABP (FABP7) has been shown to be an intracellular carrier protein that can significantly potentiate the uptake of the endocannabinoid anandamide. For this reason, there is a great interest in the discovery and development of FABP7 inhibitors for treating stress, pain, inflammation, and drug abuse. We found that in the (1) H-NMR spectrum of the protein, a well-separated downfield resonance arising from the hydrogen-bonded His93 side chain is very sensitive to ligand binding. Using this characteristic spectral marker together with another well-resolved upfield resonance from the side chain of Val84, we have identified that an adipocyte FABP (FABP4) inhibitor BMS309403 also binds tightly to FABP7. Our data demonstrated that this unique His93 downfield resonance can be used as a sensitive probe for rapidly and unambiguously identifying novel high-affinity FABP7 ligands. The findings should help accelerate the discovery of potential drug leads for the modulation of endocannabinoid transport.

Conformational study of lipophilic ligands in phospholipid model membrane systems by solution NMR.

Phospholipid bicelles were employed as a membrane bilayer model in the conformational studies of two lipophilic cannabinoids, delta(8)-THC and its O-methyl ether analogue, Me-Delta(8)-THC using conventional high-resolution NMR. A preparation of 8% (w/v) phospholipid concentration and a high DMPC/DHPC ratio (q = 2.0) was found to be optimal for not only effectively incorporating our ligands, but also providing a more bilayerlike environment suitable for conformational studies. While the conformational differences between the two cannabinoids could not be observed in chloroform and were barely detectable in SDS micelle solution, there is an increasing preference for the pentyl tail of Delta(8)-THC to bend toward the tricyclic ring system with increasing proportions of DMPC in the bicelle preparation. Our results highlight the advantages of exploring the conformational properties of cannabinoids using bicelle preparations as a medium that more closely resembles biological membrane bilayers and eliminates the need for isotopic labeling. This approach should also be of more general value for studying the interactions of other cannabinoids and biologically active, hydrophobic or amphipathic, small molecules with membranes.

The interaction of fatty acid amide hydrolase (FAAH) inhibitors with an anandamide carrier protein using (19)F-NMR.

It has been reported that the endocannabinoid anandamide (AEA) binds to a class of fatty acid-binding proteins and serum albumin which can serve as carrier proteins and potentiate the cellular uptake of AEA and its intracellular translocation. Here, we employed (19)F nuclear magnetic resonance spectroscopy to study the interactions of serum albumin with two inhibitors of fatty acid amide hydrolase (FAAH), the enzyme involved in the deactivation of anandamide. We found that, for both inhibitors AM5206 and AM5207, the primary binding site on serum albumin is drug site 1 located at subdomain IIA. Neither inhibitor binds to drug site 2. While AM5207 binds exclusively to drug site 1, AM5206 also interacts with other fatty acid-binding sites on serum albumin. Additionally, AM5206 has an affinity for serum albumin approximately one order of magnitude higher than that of AM5207. The data suggest that interactions of FAAH inhibitors with albumin may provide added advantages for their ability to modulate endocannabinoid levels for a range of applications including analgesia, antiemesis, and neuroprotection.

Targeting the Endocannabinoid System for Neuroprotection: A 19F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA.

Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using 19F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

[Gastroscopic and pathological characteristics of patients with or without schistosomiasis in Poyang Lake area].

OBJECTIVES: To understand the gastroscopic and pathological characteristics of patients with miasis in Poyang Lake area, and to explore the relationship between schistosomiasis and pathological changes of gastric mucosa. METHODS: Volunteers with or without schistosomiasis were recruited and divided into a case group and a control group. All the objects were examined by electronic gastroscopy and pathological examinations. RESULTS: Two hundred and fifty-three volunteers diagnosed with chronic or advanced schistosomiasis in the case group showed different degrees of gastric mucosal changes, including 7 cases with schistosomal eggs deposited beneath the gastric mucosa (with an incidence of 2.77%) , 33 cases with dysplastic hyperplasia and intestinal metaplasia (with an incidence of precancerous lesion of 13.04%), and 1 case with gastric cancer. While in the 200 volunteers without schistosomiasis in the control group, the results showed milder gastric mucosal changes, 33 cases were detected with dysplastic hyperplasia and intestinal metaplasia (with an incidence of 7.50%), and 1 case was diagnosed gastric cancer. The difference between the incidences of precancerous lesion in the two groups had no statistic significance (P > 0.05). CONCLUSIONS: The incidence and extent of gastric mucosal changes in schistosomiasis patients are higher and more serious than those in non-schistosomiasis patients, and gastrointestinal schistosomiasis is not related to gastric cancer.

Magnetically aligned bicelles to study the orientation of lipophilic ligands in membrane bilayers.

Magnetically aligned bicelles were used as a model membrane to study the orientation and dynamic properties of two cannabinoids (Delta (8)-THC and Me-Delta (8)-THC) using (31)P and (2)H NMR. The uniform alignment of the bicelles allowed us to obtain well resolved deuterium spectra from a solution NMR spectrometer. The preferred orientations of Delta (8)-THC and Me-Delta (8)-THC were calculated on the basis of the measurements of individual quadrupolar splittings. Our results agree with previous experiments using multilamellar membranes as well as with molecular dynamics simulation data described here. In conjunction with our earlier report using small and fast tumbling bicelles, the present work of well aligned bicelles shows that bicelle preparations can provide either pseudoisotropic or anisotropic NMR spectra to study the conformation, orientation, and dynamic properties of ligands in membrane bilayers. Such data are of critical value for understanding the interactions of lipophilic drug molecules with membrane proteins.

The conformation, location, and dynamic properties of the endocannabinoid ligand anandamide in a membrane bilayer.

The endogenous cannabinoid ligand anandamide is biosynthesized from membrane phospholipid precursors and is believed to reach its sites of action on the CB1 and CB2 receptors through fast lateral diffusion within the cell membrane. To gain a better insight on the stereochemical features of its association with the cell membrane and its interaction with the cannabinoid receptors, we have studied its conformation, location, and dynamic properties in a dipalmitoylphosphatidylcholine multilamellar model membrane bilayer system. By exploiting the bilayer lattice as an internal three-dimensional reference grid, the conformation and location of anandamide were determined by measuring selected inter- and intramolecular distances between strategically introduced isotopic labels using the rotational echo double resonance (REDOR) NMR method. A molecular model was proposed to represent the structural features of our anandamide/lipid system and was subsequently used in calculating the multispin dephasing curves. Our results demonstrate that anandamide adopts an extended conformation within the membrane with its headgroup at the level of the phospholipid polar group and its terminal methyl group near the bilayer center. Parallel static (2)H NMR experiments further confirmed these findings and provided evidence that anandamide experiences dynamic properties similar to those of the membrane phospholipids and produces no perturbation to the bilayer. Our results are congruent with a hypothesis that anandamide approaches its binding site by laterally diffusing within one membrane leaflet in an extended conformation and interacts with a hydrophobic groove formed by helices 3 and 6 of CB1, where its terminal carbon is positioned close to a key cysteine residue in helix 6 leading to receptor activation.