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A venus flytrap mechanism for activation and desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors.

Desensitization of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptor channels is an important process shaping the time course of synaptic excitation. Upon desensitization, the receptor channel closes and the agonist affinity increases. So far, the nature of the structural rearrangements leading to these events was unknown. On the basis of the structural homology of the ligand binding domains of AMPA receptors and of the bilobated bacterial periplasmic proteins, we now show that agonist interaction with one lobe of the GluR1 subunit of homomeric AMPA receptors controls channel activation while additional interactions with the other lobe cause channel desensitization. Accordingly, we suggest that the transition of the AMPA receptor channel to the desensitized state involves the agonist-mediated stabilization of the closed lobe conformation of its binding domain and is a process akin to that used by the venus flytrap.

Animals↗

Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding.

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.

Amino Acid Sequence↗

The Venus's-flytrap and cysteine-rich domains of the human Ca2+ receptor are not linked by disulfide bonds.

The extracellular N-terminal domain of the human Ca(2+) receptor (hCaR) consists of a Venus's-flytrap (VFT) domain and a cysteine-rich (Cys-rich) domain. We have shown earlier that the Cys-rich domain is critical for signal transmission from the VFT domain to the seven-transmembrane domain. The VFT domain contains 10 cysteines: two of them (Cys(129) and Cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (Cys(60)-Cys(101), Cys(358)-Cys(395), and Cys(437)-Cys(449)) are predicted to form three intramolecular disulfide bonds. The Cys-rich domain contains nine cysteines, the involvement of which in disulfide bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys(236) and Cys(482), form disulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site inserted between the VFT domain and the Cys-rich domain. These mutant hCaRs remain fully functional compared with the wild type hCaR. After TEV protease digestion of the mutant hCaR proteins, dimers of the VFT were identified on Western blot under nonreducing conditions. We concluded that there is no disulfide bond between the VFT and the Cys-rich domains in the hCaR.

Amino Acid Sequence↗

The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing.

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.

Amino Acids↗

Delineating a Ca2+ binding pocket within the venus flytrap module of the human calcium-sensing receptor.

The Ca(2+)-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca(2+) recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca(2+) binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca(2+)-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca(2+) binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca(2+) homeostasis. Altogether, these data define a Ca(2+) binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca(2+) sensing by these receptors.

Amino Acid Sequence↗

Isoflavone intake in four different European countries: the VENUS approach.

The aim of this study was to identify the level of isoflavone intake (total isoflavones, daidzein and genistein) in four European countries: Ireland, Italy, The Netherlands and the UK. For this purpose national food composition databases of isoflavone content were created in a comparable way, using the Vegetal Estrogens in Nutrition and the Skeleton (VENUS) analytical database as a common basis, and appropriate food consumption data were selected. The isoflavone intake in Ireland, Italy, The Netherlands and the UK is on average less than 1 mg/d. Small groups of consumers of soya foods could be identified in Ireland, The Netherlands and the UK. The estimated intake levels are low compared with those found in typical Asian diets (approximately 20-100 mg/d) and also low compared with levels where physiological effects are expected (60-100 mg/d). The results (including a subgroup analysis of soya product consumers) showed that such levels are difficult to achieve with the European diets studied here.

Adolescent↗

Receptor response in Venus's fly-trap.

The insect-trapping movement of the plant Dionaea muscipula (Venus's fly-trap) is mediated by the stimulation of mechanosensory hairs located on the surface of the trap. It is known that stimulation of the hairs is followed by action potentials which are propagated over the surface of the trap. It has been reported that action potentials always precede trap closure. The occurrence of non-propagated receptor potentials is reported here. Receptor potentials always precede the action potentials. The receptor potential appears to couple the mechanical stimulation step to the action potential step of the preying sequence. Receptor potentials elicited by mechanical stimulation of a sensory hair were measured by using the hair as an integral part of the current-measuring path. The tip of the hair was cut off exposing the medullary tissue; this provided a natural extension of the measuring electrode into the receptor region at the base of the hair. A measuring pipette electrode was slipped over the cut tip of the hair. Positive and negative receptor potentials were measured. Evidence is presented which supports the hypothesis that the positive and negative receptor potentials originate from independent sources. An analysis is made of (a) the relation of the parameters of mechanical stimuli to the magnitude of the receptor potential, and (b) the relation of the receptor potentials to the action potential. The hypothesis that the positive receptor potential is the generator of the action potential is consistent with these data.

Electrophysiology↗

Response properties of a sensory hair excised from Venus's flytrap.

Multicellular sensory hairs were excised from the leaf of Venus's flytrap, and the sensory cells were identified by a destructive dissection technique. The sensory layer includes a radially symmetrical rosette of 20-30 apparently identical cells, and the sensory cells are organized in a plane normal to the long axis of the sensory hair. The sensory cells were probed with intracellular glass electrodes. The resting membrane potential was about -80 mv, and the response to a mechanical stimulus consisted of a graded response and an "action potential." The action potential appears to be similar to the action potential which propagates over the surface of the leaf. In the absence of stimulation, the upper and lower membranes of a single sensory cell behave in an electrically symmetrical fashion. Upon stimulation, however, the upper and lower membranes become electrically asymmetrical. Limiting values for the response asymmetry were calculated on the hypothesis of an electrical model consistent with the histology of the sensory cells.

Action Potentials↗

Evaluation of the neutron spectrum and dose assessment around the venus reactor.

An assessment of the neutron field near the VENUS reactor is made in order to evaluate the neutron dose to the operators, particularly in an area near the reactor shielding and in the control room. Therefore, a full MCNPX model of the shielding geometry was developed. The source term used in the simulation is derived from a criticality calculation done beforehand. Calculations are compared to routine neutron dose rate measurements and show good agreement. The MCNPX model developed easily allows core adaptations in order to evaluate the effect of future core configuration on the neutron dose to the operators.

Algorithms↗

Mars and Venus: unequal planets.

Self-help books, a pervasive and influential aspect of society, can have a beneficial or detrimental effect on the therapeutic process. This article describes a thematic analysis and feminist critique of the best-selling self-help book, Men are from Mars, Women are from Venus. This analysis revealed that the author's materials are inconsistent with significant family therapy research findings and key principles of feminist theories. His descriptions of each gender and his recommendations for improving relationships serve to endorse and encourage power differentials between women and men.

Books↗

The Venus flytrap of periplasmic binding proteins: an ancient protein module present in multiple drug receptors.

Located between the inner and outer membranes of Gram-negative bacteria, periplasmic binding proteins (PBPs) scavenge or sense diverse nutrients in the environment by coupling to transporters or chemotaxis receptors in the inner membrane. Their three-dimensional structures have been deduced in atomic detail with the use of X-ray crystallography, both in the free and liganded state. PBPs consist of two large lobes that close around the bound ligand, resembling a Venus flytrap. This architecture is reiterated in transcriptional regulators, such as the lac repressors. In the process of evolution, genes encoding the PBPs have fused with genes for integral membrane proteins. Thus, diverse mammalian receptors contain extracellular ligand binding domains that are homologous to the PBPs; these include glutamate/glycine-gated ion channels such as the NMDA receptor, G protein-coupled receptors, including metabotropic glutamate, GABA-B, calcium sensing, and pheromone receptors, and atrial natriuretic peptide-guanylate cyclase receptors. Many of these receptors are promising drug targets. On the basis of homology to PBPs and a recently resolved crystal structure of the extracellular binding domain of a glutamate receptor ion channel, it is possible to construct three-dimensional models of their ligand binding domains. Together with the extensive information available on the mechanism of ligand binding to PBPs, such models can serve as a guide in drug discovery.

Animals↗

Metallothionein levels in the bivalves Callista chione and Venus verrucosa from two Mediterranean sites.

Metallothioneins levels (MTs) in the clams Callista chione and Venus verrucosa, collected from two coastal sites in Greece, were determined and quantified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and a spectrophotometric assay (Ellman's reaction). SDS-PAGE separation in the digestive gland, which represents the hepato-pancreas in clams, demonstrated the presence of MTs similar to mammalian MT (rabbit liver Cd, Zn-thionein). No other SH-containing proteins apart from the MTs were detected. MT levels quantified by the Ellman's reaction indicated seasonal variation for both species. The highest values were recorded in the spring and the lowest in the autumn. The seasonal variation and the differences in the MT levels of the two areas seem to be related to the reproductive cycle of the organisms as well as to abiotic factors of each area. Our results show that both C. chione and V. verrucosa have the potential to be used as biomarkers of metal pollution, provided that the influence of the external factors is safely quantified.

Animals↗

Monitoring of infections by protozoa of the genera Nematopsis, Perkinsus and Porospora in the smooth venus clam Callista chione from the North-Western Adriatic Sea (Italy).

Marketable smooth venus clams Callista chione from natural banks of Chioggia (Venice) and Goro (Ferrara), North-Western Adriatic Sea (Italy), were examined for protozoan parasites from November 1996 to November 1998. Out of the 375 bivalves examined, 149 (39.7%) were infected by Nematopsis sp. and 325 (86.7%) by Porospora sp. Oocysts of Nematopsis sp. were present with a prevalence that varied from 100% in November 1996 to 5% in June 1998; cystic and naked sporozoites of Porospora sp. were very common, with a prevalence of 100%. Out of the 229 bivalves examined between January and November 1998, 63 (27.5%) were also infected by Perkinsus sp.; the prevalence of Perkinsus sp. varied from 9.1% in January to 50% in February. To our knowledge this is the first report of Porospora sp. and Perkinsus sp. in C. chione.

Animals↗

Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina.

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.

Animals↗

[A network of epidemiological information in community medicine practice: methodological principles of the VENUS Program].

Implementation of epidemiological studies involving the participation of large networks of ambulatory care physicians requires prior resolution of the methodological dilemma between a "volunteer sentinel doctors" approach and one based on sampling techniques. A study of the VENUS program (concerning Sexually Transmitted Diseases) illustrates this latter approach for which formulae are proposed for estimating the rate of incidence in the population. Methodological implications of this technique are commented upon, and the complementary indications for the two techniques for gathering information on ambulatory morbidity are discussed.

Ambulatory Care↗

[Effect of cadmium on the activity of alkaline phosphatase (3.1.3.1) of Venus gallina].

The marine mollusc Venus gallina was exposed to 32 days sublethal concentrations of Cadmium (0.1 microliter/ml). The activity of alkaline phosphatase was assayed every four days. In the controls the activity was also tested in different tissues and in the soft tissue for the pH dependence. Moreover the influence of direct addition of 10(-4) M Cd on enzyme preparation in vitro was assayed. From the results there are no consistent relationships between the direct in vitro effects of the metal on the enzyme, that is inhibitory, and the effect of exposing the whole animal to the same metal, in this case no inhibition was observed.

Alkaline Phosphatase↗

Pharmacokinetics of an anti-carcinoembryonic antigen monoclonal antibody conjugated to a bifunctional transition metal carborane complex (venus flytrap cluster) in tumor-bearing mice.

An anticarcinoembryonic antigen (CEA) monoclonal antibody, T84.66, has been conjugated to a metallocarborane complex (Venus flytrap cluster, VFC) containing 57Co. This radioimmunoconjugate, 57Co-VFC-T84.66, retained > 90% immunoreactivity, was stable in serum (7 days) and demonstrated good localization in LS174T tumor xenografts. Pharmacokinetics of 57Co-VFC-T84.66 in tumor-bearing mice were compared to T84.66 Mab conjugated with either DTPA or its benzylisothiocyanate derivative (BzDTPA) labeled with 111In. Whole-body half-life for VFC-T84.66 was less (t1/2 = 62 hr) than that for either DTPA-T84.66 (t1/2 = 157 hr) or BzDTPA-T84.66 (t1/2 = 167 hr). Blood clearance was similar for all three radioimmunoconjugates (t1/2 = 22 hr). Hepatic uptake of the radiolabel was rapid and remained constant for 7 days for both DTPA radioimmunoconjugates (DTPA radioimmunoconjugate = 13.7 +/- 1.5 %ID/g; BzDTPA radioimmunoconjugate = 10.4 +/- 1.7%ID/g). For VFC, however, liver radioactivity decreased from 19.1 +/- 0.6%ID/g at 1 hr to 0.9 +/- 0.1 %ID/g 7 days postinjection, suggesting a possible role for VFC radioimmunoconjugate in the imaging and therapy of liver metastases.

Animals↗