Mechanism of partial agonism at the GluR2 AMPA receptor: Measurements of lobe orientation in solution

The mechanism by which the binding of a neurotransmitter to a receptor leads to channel opening is a central issue in molecular neurobiology. The structure of the agonist binding domain of ionotropic glutamate receptors has led to an improved understanding of the changes in structure that accompany agonist binding and have provided important clues about the link between these structural changes and channel activation and desensitization. However, because the binding domain has exhibited different structures under different crystallization conditions, understanding the structure in the absence of crystal packing is of considerable importance. The orientation of the two lobes of the binding domain in the presence of a full agonist, an antagonist, and several partial agonists was measured using NMR spectroscopy by employing residual dipolar couplings. For some partial agonists, the solution conformation differs from that observed in the crystal. A model of channel activation based on the results is discussed.     

amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis

Background  

The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation.     

Specific amplification of the 18S rRNA gene as a method to detect zebra mussel (Dreissena polymorpha) larvae in plankton samples

An important issue in the management of zebra mussel (Dreissena polymorpha) populations is early, rapid, and accurate detection of the planktonic larvae (veliger) of the zebra mussel. The goal of this study was to explore the feasibility of developing a molecular approach for the detection of zebra mussel larvae in diverse environments. In this study a Dreissena polymorpha-specific 18S ribosomal RNA gene targeted oligonucleotide primer (ZEB-715a) and Polymerase Chain Reaction (PCR) assay was developed and compared with cross-polarized microscopy as a means to detect zebra mussel veligers in plankton samples. The design of the zebra mussel-specific primer was facilitated by sequencing nearly the complete 18S rRNA gene from the zebra mussel and three other closely related freshwater Veneroids including the quagga mussel (D. bugensis), the dark false mussel (Mytilopsis leucophaeata), and the Asian freshwater clam (Corbicula fluminea). The specificity of the primer for the zebra mussel was empirically tested by using the primer as a direct probe in a blot hybridization format. A single veliger in a plankton sample could be detected by PCR using this approach. Veliger detection sensitivity using the PCR approach was estimated to be over 300 times more sensitive than cross-polarized light microscopy based techniques. Cross-polarized light microscopy and the PCR technique were used to identify the presence of zebra mussel larvae in plankton samples that were collected from a variety of natural and industrial water sources. Detection results (presence or absence) were generally consistent between the two methods. Although additional studies will be required before routine application of molecular based veliger detection technology is available, a long-term goal of this work is the application of molecular technology to the development of a field device for the routine detection and quantification of zebra mussel veligers.     

Asp-225 and glu-375 in autocatalytic attachment of the prosthetic heme group of lactoperoxidase.

The heme in lactoperoxidase is attached to the protein by ester bonds between the heme 1- and 5-methyl groups and Glu-375 and Asp-275, respectively. To investigate the cross-linking process, we have examined the D225E, E375D, and D225E/E375D mutants of bovine lactoperoxidase. The heme in the E375D mutant is only partially covalently bound, but exposure to H(2)O(2) results in complete covalent binding and a fully active protein. Digestion of this mutant shows that the heme is primarily bound through its 5-methyl group. Excess H(2)O(2) increases the proportion of the doubly linked species without augmenting enzyme activity. The D225E mutant has little covalently bound heme and a much lower activity, neither of which are significantly increased by the addition of heme and H(2)O(2). The heme is linked to this protein through a single bond to the 1-methyl group. The D225E/E375D mutant has no covalently bound heme and no activity. A small amount of iron 1-hydroxymethylprotoporphyrin IX is obtained from the wild-type enzyme along with the predominant dihydroxylated derivative. The results establish that a single covalent link suffices to achieve maximum catalytic activity and suggest that the 5-hydroxymethyl bond may form before the 1-hydroxymethyl bond.     

Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.     

Insulin-like growth factor I increases alpha Vbeta 3 affinity by increasing the amount of integrin-associated protein that is associated with non-raft domains of the cellular membrane.

Insulin-like growth factor I (IGF-I) stimulates an increase in alpha(V)beta(3) ligand binding. Stimulation of smooth muscle cells by IGF-I requires alpha(V)beta(3) ligand occupancy, and enhanced alpha(V)beta(3) ligand occupancy augments IGF-I actions. Therefore, IGF-I-induced changes in alpha(V)beta(3) ligand binding may act to further enhance IGF-I actions. Integrin-associated protein (IAP) has been shown to be associated with alpha(V)beta(3) and is required for the binding of alpha(V)beta(3) to vitronectin-coated beads. We therefore investigated whether IGF-I could stimulate IAP-alpha(V)beta(3) association resulting in enhanced ligand binding. IGF-I stimulated an increase in IAP-alpha(V)beta(3) association. This was due, at least in part, to an IGF-I-stimulated redistribution of IAP from the Triton-insoluble fraction of the cell to the Triton-soluble fraction of the cell, where most of the alpha(V)beta(3) was located. Inhibition of the phosphatidylinositol 3-kinase pathway blocked both the redistribution of IAP and the increase in IAP-alpha(V)beta(3) association, providing further evidence that the redistribution of IAP is essential for the increase in association. An anti-IAP monoclonal antibody, blocked both the IGF-I-stimulated increase in IAP-alpha(V)beta(3) complex formation and cell migration. IGF-I-stimulated translocation of IAP and increase in IAP-alpha(V)beta(3) association represent an important process by which IGF-I modulates alpha(V)beta(3) ligand binding and cellular responses.