Structures of adnectin/protein complexes reveal an expanded binding footprint

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Highlights? Adnectins interact with targets using multiple binding modes ? Residues outside of the diversified loops can interact with ligand ? Some fixed scaffold residues contribute to the binding energy ? Adnectins can interact with epitopes that may be inaccessible to antibodies     

Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures

Although there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of ～1,000 genes important for neural functions by in?situ hybridization at?a cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual gene's expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, and in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain.     

Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. II. Importance of accurate toxicity measurement

In a previous publication, Fowler et al. [4] demonstrated that the seemingly high rate of false or misleading positive results obtained in in vitro cytogenesis assays for genotoxicity - when compared with in vivo genotoxicity or rodent carcinogenicity data - was greater when rodent cell lines were used that were also reported to have mutant or non-functional p53. As part of a larger project for improvement of in vitro mammalian cell assays, we have investigated the impact of different toxicity measures, commonly used in in vitro cytogenetic assays, on the occurrence of misleading positive results. From a list of 19 chemicals that produce "false" positive results in in vitro mammalian cell assays [10], six substances that had given positive responses in CHO, CHL and TK6 cells [4], were evaluated for micronucleus induction in vitro, with different measures of toxicity for selection of the top concentration. The data show that estimating toxicity by relative cell count (RCC) or replication index (RI) consistently underestimates the toxicity observed by other measures (Relative Population Doubling, RPD, or Relative Increase in Cell Count, RICC). RCC and RI are more likely to lead to selection of concentrations for micronucleus scoring that are highly cytotoxic and thus could potentially lead to artefacts of toxicity being scored (elevated levels of apoptosis and necrosis), generating misleading positive results. These results suggest that a further reduction in the frequency of misleading positive results in in vitro cytogenetic assays can be achieved with this set of chemicals, by avoiding the use of toxicity measures that underestimate the level of toxicity induced.     

Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors.     

Integrated simulation with experimentation is a powerful tool for understanding diatom valve morphogenesis

We present a number of promising examples of computational studies, which improve our understanding of the morphogenesis process of diatom cell walls. Each example considers a different physical scenario whereby computational and mathematical models are used to evaluate hypotheses pertaining to diatom valve formation; considering the roles of cytoskeletal elements, interactions between cell components that might generate patterned structures from the submicron (nanoscale) to cell level, and the effect of environmental variables. We propose that the complex, multiscale phenomenon of diatom valve morphogenesis requires better integration of computational/mathematical and experimental procedures if we are to untangle all the contributing processes. Finally we outline a plan for future directions, to achieve this integration and further the field of diatom morphogenesis research.     

Distributions of active spinal cord neurons during swimming and scratching motor patterns

The spinal cord can generate motor patterns underlying several kinds of limb movements. Many spinal interneurons are multifunctional, contributing to multiple limb movements, but others are specialized. It is unclear whether anatomical distributions of activated neurons differ for different limb movements. We examined distributions of activated neurons for locomotion and scratching using an activity-dependent dye. Adult turtles were stimulated to generate repeatedly forward swimming, rostral scratching, pocket scratching, or caudal scratching motor patterns, while sulforhodamine 101 was applied to the spinal cord. Sulforhodamine-labeled neurons were widely distributed rostrocaudally, dorsoventrally, and mediolaterally after each motor pattern, concentrated bilaterally in the deep dorsal horn, the lateral intermediate zone, and the dorsal to middle ventral horn. Labeled neurons were common in all hindlimb enlargement segments and the pre-enlargement segment following swimming and scratching, but a significantly higher percentage were in the rostral segments following swimming than rostral scratching. These findings suggest that largely the same spinal regions are activated during swimming and scratching, but there are some differences that may indicate locations of behaviorally specialized neurons. Finally, the substantial inter-animal variability following a single kind of motor pattern may indicate that essentially the same motor output is generated by anatomically variable networks.     

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.     

Turning receptors on and off with intracellular pepducins: new insights into G-protein-coupled receptor drug development

G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR1, PAR2, and PAR4), chemokine receptors (CXCR1, CXCR2, and CXCR4), sphingosine 1-phosphate receptor-3 (S1P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gα(q/11/o/13)), muscarinic acetylcholine receptor and vanilloid (TRPV1) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets.     

Probing the Structural Basis of Zn2+ Regulation of the Epithelial Na+ Channel

Extracellular Zn²⁺ activates the epithelial Na⁺ channel (ENaC) by relieving Na⁺ self-inhibition. However, a biphasic Zn²⁺ dose response was observed, suggesting that Zn²⁺ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn²⁺, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn²⁺ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn²⁺ activating site, γH239A diminished Na⁺ self-inhibition and thus concealed the activating effects of Zn²⁺. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn²⁺ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp⁵¹⁶ was predicted to be in close proximity to γHis⁸⁸. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn²⁺. Our results suggest that external Zn²⁺ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His¹⁹³, His²⁰⁰, and His²⁰² and (ii) a low-affinity Zn²⁺ inhibitory site within the palm subdomain that includes His⁸⁸ and Asp⁵¹⁶.