In Drosophila photoreceptors the multivalent PDZ protein INAD organizes the phototransduction cascade into a macromolecular signaling complex containing the effector PLC, the light-activated TRP channels, and a regulatory PKC. Previously, we showed that the subcellular localization of INAD signaling complexes is critical for signaling. Now we have examined how INAD complexes are anchored and assembled in photoreceptor cells. We find that trp mutants, or transgenic flies expressing inaD alleles that disrupt the interaction between INAD and TRP, cause the mislocalization of the entire transduction complex. The INAD-TRP interaction is not required for targeting but rather for anchoring of complexes, because INAD and TRP can be targeted independently of each other. We also show that, in addition to its scaffold role, INAD functions to preassemble transduction complexes. Preassembly of signaling complexes helps to ensure that transduction complexes with the appropriate composition end up in the proper location. This may be a general mechanism used by cells to target different signaling machinery to the pertinent subcellular location.

We have prepared ionic liquids by mixing either iron(II) chloride or iron(III) chloride with 1-butyl-3-methylimidazolium chloride (BMIC). Iron(II) chloride forms ionic liquids from a mole ratio of 1 FeCl(2)/3 BMIC to almost 1 FeCl(2)/1 BMIC. Both Raman scattering and ab initio calculations indicate that FeCl(4)(2-) is the predominant iron-containing species in these liquids. Iron(III) chloride forms ionic liquids from a mole ratio of 1 FeCl(3)/1.9 BMIC to 1.7 FeCl(3)/1 BMIC. When BMIC is in excess, Raman scattering indicates the presence of FeCl(4-). When FeCl(3) is in excess, Fe(2)Cl(7-) begins to appear and the amount of Fe(2)Cl(7-) increases with increasing amounts of FeCl(3). Ionic liquids were also prepared from a mixture of FeCl(2) and FeCl(3) and are discussed. Finally, we have used both Hartree-Fock and density functional theory methods to compute the optimized structures and vibrational spectra for these species. An analysis of the results using an all-electron basis set, 6-31G, as well as two different effective core potential basis sets, LANL2DZ and CEP-31G is presented.

A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum has been determined to 2.8-Å resolution. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe3S4] and not [Fe4S4]. The mononuclear site and the four-metal cluster are bridged by means of Cys531 and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe4S4] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an α/β (Rossmann-like) domain in the middle, and an α/β (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe4S4] cluster and another [Fe4S4] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems.

Malaria and human immunodeficiency virus (HIV) coinfections are common in pregnant women in sub-Saharan Africa. The current study shows that placentas of malaria-infected women contain 3 times as much CC chemokine receptor 5 (CCR5) RNA as placentas of women without malaria. By immunohistochemistry, CCR5(+) maternal macrophages were seen in placentas from malaria-infected women but not in placentas from malaria-uninfected women. In addition, CCR5 also was found on fetal Hofbauer cells in placentas from both groups. Thus, malaria infections increase the potential reservoir for HIV in the placenta by increasing the number of HIV target cells.

The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery.

Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from an approximately 100-fold improvement (7.4 nM) to an approximately two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).

The use of chronically implanted electrodes for neural recordings in small, freely behaving animals poses several unique technical challenges. Because of the need for an extremely lightweight apparatus, chronic recording technology has been limited to manually operated microdrives, despite the advantage of motorized manipulators for positioning electrodes. Here we describe a motorized, miniature chronically implantable microdrive for independently positioning three electrodes in the brain. The electrodes are controlled remotely, avoiding the need to disturb the animal during electrode positioning. The microdrive is approximately 6 mm in diameter, 17 mm high and weighs only 1.5 g, including the headstage preamplifier. Use of the motorized microdrive has produced a ten-fold increase in our data yield compared to those experiments done using a manually operated drive. In addition, we are able to record from multiple single neurons in the behaving animal with signal quality comparable to that seen in a head-fixed anesthetized animal. We also describe a motorized commutator that actively tracks animal rotation based on a measurement of torque in the tether.

BACKGROUND: Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator.

RESULTS: MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction.

CONCLUSIONS: MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

The stomatogastric ganglion (STG) of the crab Cancer productus contains approximately 30 neurons arrayed into two different networks (gastric mill and pyloric), each of which produces a distinct motor pattern in vitro. Here we show that the functional division of the STG into these two networks requires intact NO-cGMP signaling. Multiple nitric oxide synthase (NOS)-like proteins are expressed in the stomatogastric nervous system, and NO appears to be released as an orthograde transmitter from descending inputs to the STG. The receptor of NO, a soluble guanylate cyclase (sGC), is expressed in a subset of neurons in both motor networks. When NO diffusion or sGC activation are blocked within the ganglion, the two networks combine into a single conjoint circuit. The gastric mill motor rhythm breaks down, and several gastric neurons pattern switch and begin firing in pyloric time. The functional reorganization of the STG is both rapid and reversible, and the gastric mill motor rhythm is restored when the ganglion is returned to normal saline. Finally, pharmacological manipulations of the NO-cGMP pathway are ineffective when descending modulatory inputs to the STG are blocked. This suggests that the NO-cGMP pathway may interact with other biochemical cascades to partition rhythmic motor output from the ganglion.

In the visual cortex of many mammals, orientation preference changes smoothly along the cortical surface, with the exception of singularities such as pinwheels and fractures. The reason for the existence of these singularities has remained elusive, suggesting that they are developmental artifacts. We show that singularities reduce the length of intracortical neuronal connections for some connection rules. Therefore, pinwheels and fractures could be evolutionary adaptations keeping cortical volume to a minimum. Wire length minimization approach suggests that interspecies differences in orientation preference maps reflect differences in intracortical neuronal circuits, thus leading to experimentally testable predictions. We discuss application of our model to direction preference maps.