Scanning mutagenesis studies of the M1 muscarinic acetylcholine receptor

Following the solution of the structure of bovine rhodopsin by X-ray crystallography, it has been possible to build an improved homology model of the M(1) muscarinic acetylcholine receptor. This has been used to interpret the outcome of an extensive series of scanning and point mutagenesis studies on the transmembrane domain of the receptor. Potential intramolecular interactions enhancing the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N-methylscopolamine, and the agonist, acetylcholine have been mapped. The positively charged headgroups of these ligands appear to bind in a charge-stabilized aromatic cage formed by amino acid side chains in transmembrane (TM) helices 3, 6, and 7, while residues in TM 4 may participate in a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may help to transduce binding energy into receptor activation, possibly disrupting a set of Van der Waals interactions between a set of residues underlying the binding site which help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for activation.     

G-protein coupled receptors as allosteric machines.

Allosterism, whereby small molecule ligands produce global changes in the conformations of receptors, is a powerful mechanism for drug effect. This is illustrated by the recent data describing CCR5 antagonists as blockers of HIV infection. Allosteric effects are described in terms of a change in the tertiary conformation of the receptor. This paper outlines some unique features of allosteric antagonists as new drug entities. These include the fact that allosteric ligands have texture in antagonism (not all allosterically blocked receptors are alike), allosteric blockade is probe dependent (not all agonists and radioligands are blocked equally), and the fact that allosteric binding involves a separate site on the receptor may have relevance to duration of effect and selectivity. Dissociation between receptor function and binding also can be encountered with allosteric ligands.     

Cloning and functional characterization of a Caenorhabditis elegans muscarinic acetylcholine receptor

A cDNA clone encoding a muscarinic acetylcholine receptor (mAChR) has been isolated from the nematode Caenorhabditis elegans. The nematode mAChR, consisted of 585 amino acids, displays a high degree of amino acid sequence homology to other invertebrate and vertebrate mAChRs. Excluding a highly variable middle portion of the third intracellular loop, the C. elegans mAChR shares about 51% amino acid sequence identity with a Drosophila mAChR and 42-44% identity with human m1-m5 mAChR subtypes. Comparison of the cDNA sequence with the corresponding genomic sequence reveals that the C. elegans mAChR gene contains ten introns, eight of them in the coding region. Pharmacological profiles of the C. elegans mAChR expressed in Chinese hamster ovary (CHO) cells were shown to be similar to those of mammalian counterparts, indicating that ligand binding domains of the receptor have been conserved during evolution. When this cloned receptor was expressed in Xenopus oocytes, acetylcholine evoked a transient Cl- current. Furthermore, activation of the receptor with oxotremorine, acetylcholine or carbachol resulted in the stimulation of phosphatidylinositol metabolism in CHO cells, suggesting that the receptor is coupled to phospholipase C activation.     

Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells

Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.     

M1-M5 muscarinic receptor knockout mice as novel tools to study the physiological roles of the muscarinic cholinergic system

A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.     

Resolving revolutions: imaging and mapping by modern radar

High-performance synthetic aperture radars (SARs) for mapping demand massive digital signal processing powers. The fall in the cost of computing devices has recently passed the point at which such processors can be afforded and SARs are now being used in a range of applications. The analogous inverse synthetic aperture radar (ISAR), which enables moving targets to be imaged by stationary or moving radars, is also becoming widely used. The unifying principle underlying SARs and ISARs is presented and the common parameters defining the performances of both types of radar are derived. A novel technique is described which enables the radar to measure the random angular spin of a ship at sea, thereby permitting it to be imaged deterministically by ISAR. Results are presented from a representative selection of ISARs and SARs ranging from the imaging of model targets by ISARs operating at scaled-up frequencies through to the mapping of the surface of Venus by a satellite SAR. The paper concludes with a review of likely future developments of these types of radar and suggests that further major advances are possible     

Conserved sequence motifs of olfactory receptor-like proteins may participate in upstream and downstream signal transduction.

Olfactory receptor-like proteins (ORLs) are seven transmembrane domain G protein-coupled receptors. We hypothesize that, in contrast with the hypervariable regions that may interact with a variety of odor ligands, the external and internal segments of the ORLs contain conserved regions that may interact with conserved olfactory binding proteins or direct axon guidance, and with G proteins, respectively. To test this hypothesis, a comprehensive analysis using the multiple expectation maximization for motif elicitation discovery tool was performed in all the full-length ORL clones deposited in the public section of the olfactory receptor database. Ten motifs have been identified that are present in all the olfactory receptors, in the same order, and are not present in other G protein-coupled receptors. These motifs are concentrated either in the extracellular-most or the intracellular-most parts of the receptors. The generality of these motifs was verified by their existence in the partial ORLs and 28 newly identified human receptors. The existence and localization of these motifs, suggest that they may be involved in the interactions of the receptors with their upstream and downstream signaling partners. In addition, the motifs present an additional to overall homology criterion for ORL family definition.     

Pharmacological analysis of the contractile role of M2 and M3 muscarinic receptors in smooth muscle

Muscarinic receptors expressed on smooth muscle cells are primarily of the M(2) and M(3) subtypes. The M(3) subtype triggers contraction through an interaction with G(q) proteins to stimulate phosphoinositide hydrolysis and mobilize Ca(2+). In contrast, activation of M(2) receptors modulates contraction by preventing relaxation or by potentiating M(3) receptor-mediated contractions, which enhances heterologous desensitization. These effects can be explained by the coupling of M(2) receptors to G(i) proteins that mediate an inhibition of adenylyl cyclase and calcium-activated potassium channels. The pharmacological antagonism of a response mediated through an interaction between M(2) and M(3) receptors has been shown to resemble the profile of the directly acting receptor (M(3)), primarily, and not that of the conditional receptor (M(2)). Evidence for a contractile role of the M(2) receptor has been obtained by inactivating its signaling pathway with pertussis toxin or by measuring contractile effects of muscarinic agonists after M(3) receptors have been covalently inactivated. Under these conditions, M(2) receptors have been shown to mediate an inhibition of the relaxant effects of agents, like isoproterenol, on the contractile effects of nonmuscarinic spasmogens. Muscarinic M(2) and M(3) receptor knockout mice are useful tools for exploring interactions between these receptors in smooth muscle.     

Specific absorption rates in a flat phantom in the near-field of dipole antennas

National and international regulatory bodies require compliance testing procedures for hand-held wireless telephones. The IEEE has promulgated a compliance verification procedure (P1528) to be followed in verifying whether a wireless phone is compliant with international standards. Manufacturers are required to assess the maximum near-field exposures that phones might produce in the head of a user. A recent intercomparison of the testing procedure has involved the cooperation of 15 government and industrial laboratories. These laboratories measured the 1- and 10-cm/sup 3/ cubic volume-averaged specific absorption rates (SARs) in a flat phantom filled with a standardized lossy dielectric fluid. The phantom absorber was placed in the near field of a custom dipole antenna. In support of this effort, we have performed a theoretical analysis of the expected SARs in the measurement system, which has allowed comparison with experiment. We have also been able to compare the 1and 10-cm/sup 3/ volume-averaged SARs for cubic and maximum SAR volumes. There is generally good agreement between experimental and theoretical SAR spatial patterns, and SARs averaged over 1- and 10-cm/sup 3/ cubic volumes. The 1- and 10-cm/sup 3/ average SARs in the shapes giving maximum SARs are about 35% larger compared to cubic volumes.     

Calculation of EM power deposition for operator exposure to RFinduction heaters

Calculations are presented for electromagnetic power deposition (specific absorption rates, or SARs) in an anatomically realistic model of the human torso for spatially varying leakage magnetic fields due to a 450 kHz RF induction heater. A previously described impedance method that neglects the effects of the operator's body on the source, because of weak coupling, is used for the calculations. SARs on the order of a few μW/Kg are found for magnetic fields on the order of 1.8 A/m at the central plane of the model. This implies that fairly low SARs result from leakage magnetic fields on the order of 1-20 A/m that are typical of RF induction heaters     

Specific absorption rates (SARs) induced in head tissues by microwave radiation from cell phones

The cellular/mobile phone industry has been reluctant to disclose SAR values to the public in the past. The decision by the Cellular Telecommunication Industry Association (CTIA) to require manufacturers to disclose SARs emerged after it became clear that SAR information was going to appear on the FCC Web site in a user-friendly form. It was also prompted by a British government panel's call, on May 11, 2000, for the SAR of each mobile phone to be printed on the box in which the phone is sold. The wireless telephone industry is concerned about how the public may use the SARs, because, according to one industry source, "SARs by themselves may be misleading and variations in SARs do not represent a variation in safety." Indeed, SAR values can vary as a function of the carrier frequency-for example, 850 or 1900 MHz, used to transmit the telephone message-or how the handset is held. The article presents some insights into SAR, what it is, how is it determined, and what it means.     

THE RELATIONSHIP BETWEEN THE EM ENERGY ABSORPTION OF HUMAN BODY AND THE INCIDENT DIRECTION AND POLARIZATION OF PLANE WAVE

The problems of EM energy absorption of human body irradiated by plane wave arediscussed by the Finite-Difference Time-Domain(FD-TD)method.The local Specific AbsorptionRates(SARs),the whole-body average SARs and the layer average SARs for the inhomogeneousblock model of human body with different incident direction and different polarization of theincident waves are calculated.The results show that the appearance of maximum EM energyabsorption is not always at the situation of the front incidence and the local SARs are moreimportant for the interaction of the EM fields with human body.All results provide more infor-mation about the electromagnetic dosimetry for human body.     

Electromagnetic absorption in the human head from experimental6-GHz handheld transceivers

We have used a new millimeter-resolution MRI-based model of the human body to calculate the electromagnetic absorption in the head and neck for three experimental Yagi antennas suggested for handheld transceivers of a proposed 6-GHz personal communication network (PCN) system. The SAR distributions are obtained with a resolution of 1.974×1.974×1.5 mm for transceivers that are held against the ears and tilted forward by 33°. The finite-difference time-domain technique is used to calculate the EM fields and SARs for the transceiver, antenna, and head and neck coupled region that is divided into 158×84×188 or nearly 2.5 million cells. The highlights of the numerical calculations are verified by means of a head-shaped experimental model made of tissue-equivalent materials simulating the electrical properties (ϵ_r,σ) of the skull, brain, muscle, eyes, and ears developed for use at 6 GHz. Because of the proximity to the antenna, the highest SARs are obtained for the upper part of the ear. For a planned radiated power of 0.6 W, the peak SARs averaged over any 1 g of tissue defined as a tissue volume in the shape of a cube are on the order of 0.5-1.0 W/kg for two of the proposed antennas and considerably higher (2.06 W/kg) for the third antenna using a narrower off-axis reflector. Low SARs for the first two antennas are likely due to the shielding provided by the relatively wider strip reflector plates used for these antennas     

Molecular modelling of the 5-hydroxytryptamine receptors.

Molecular models of the 5-hydroxytryptamine receptors are reviewed with particular emphasis upon ligand receptor interactions. The use of such models in the areas of drug design and the design and rationalisation of experiments is discussed. It is clear that many groups have modelled the 5-HT receptors using either the bacteriorhodopsin structure, rhodopsin electron density map or rhodopsin alpha-carbon template. Although it is difficult to assess the accuracy of these models it is expected that the rhodopsin based models show improved accuracy over the bacteriorhodopsin based models. Nevertheless models that are thoroughly validated with experimental mutagenesis and ligand binding data are useful in a qualitative sense providing ideas upon target compounds to synthesise and the design and rationalisation of mutagenesis experiments.     

The monoaminergic receptors of the internal organs in rats with a tumor and after partial hepatectomy

Sensitivity to phenylephrine, isoproterenol, serotonin, oxytocin, acetylcholine and barium chloride of vas deferens uterus und fundus strip was studied comparatively in hepatectomized and sarcoma-45, sarcoma-M1, Walker carcinosarcoma and Zajdela ascites hepatoma bearing rats. The contractile response to monoamines and oxytocin was considerably lower or absent at certain periods after hepatectomy or tumour grafting. Effects of biogenic amine antagonists were also substantially altered. The response to isoproterenol, acetylcholine and barium chloride remained unchanged. Apparently a selective alteration of a response of visceral smooth muscles mediated through alpha-adrenergic and D-serotonin receptors occurred not only during the tumour growth but also in the case of active (extensive) proliferation of the normal tissue.     

Cellular signaling mechanisms for muscarinic acetylcholine receptors

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.     

Elastic Modulus Dependence on the Specific Adhesion of Hydrogels

Mechanosensitivity in biology, e.g., cells responding to material stiffness, is important for the design of synthetic biomaterials. It is caused by protein receptors able to undergo conformational changes depending on mechanical stress during adhesion processes. Here the elastic modulus dependence of adhesive interactions is systematically quantified using ligand–receptor model systems that are generally not thought to be mechanosensitive: biotin–avidin, mannose–concanavalin A, and electrostatic interactions between carboxylic acids and polycationic surfaces. Interactions are measured by microgel sensors of different stiffness adhering to surfaces presenting a corresponding binding partner. Adhesion is generally decreased for softer microgels due to reduced density of binding partners. Density‐normalized data show that low‐affinity carbohydrate ligands exhibit reduced binding in softer networks, probably due to increased network conformational entropy. However, in case of stronger interactions with large interaction range (electrostatic) and large lifetime (biotin–avidin) density normalized adhesion is increased. This suggests compensation of entropic repulsion for softer networks probably due to their increased mechanical deformation upon microgel adhesion and enhanced cooperative binding. In essence, experiments indicate that soft interacting polymer materials exhibit entropic repulsion, which can be overcome by strongly interacting species in the network harnessing network flexibility in order to increase adhesion.     

SAR and induced current distributions for operator exposure to RFdielectric sealers

The finite-difference time-domain method is used to calculate local, layer-averaged, and whole-body averaged specific absorption rates (SARs) and induced current distributions in a 16-tissue, anatomically based, 5628-cell model of a human to assess operator exposure to RF sealers. Industrially relevant shapes and dimensions of commonly used RF dielectric heaters using parallel-plate and bar-type electrodes are considered. Realistic postures of the human operators are used for the calculations, including extending arms to simulate working conditions or an operator sitting on a wooden or metallic stool. Due to the high-intensity leakage fields in proximity to the RF applicators, some of the highest induced currents and SARs are calculated for the hands and the ankles and, in the sitting position, the knees. It is concluded that steps should therefore be taken either to reduce the leakage fields or shield the hands and the knees if it is necessary for them to be in high leakage field regions     

噪声干扰环境下SAR的目标检测能力分析

SAR的目标检测是指从SAR图像的背景杂波和干扰中检测目标，检测概率是衡量SAR目标检测能力的重要指标。通过计算噪声干扰环境下SAR的目标检测概率可以定量描述SAR的目标检测能力在噪声干扰环境下受削弱的程度。基于恒虚警检测技术，本文给出噪声干扰环境下SAR目标检测概率的计算方法，并对噪声干扰环境下SAR的目标检测能力进行了分析。