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1.  Randomized controlled trial of trigeminal nerve stimulation for drug-resistant epilepsy 
Neurology  2013;80(9):786-791.
Objective:
To explore the safety and efficacy of external trigeminal nerve stimulation (eTNS) in patients with drug-resistant epilepsy (DRE) using a double-blind randomized controlled trial design, and to test the suitability of treatment and control parameters in preparation for a phase III multicenter clinical trial.
Methods:
This is a double-blind randomized active-control trial in DRE. Fifty subjects with 2 or more partial onset seizures per month (complex partial or tonic-clonic) entered a 6-week baseline period, and then were evaluated at 6, 12, and 18 weeks during the acute treatment period. Subjects were randomized to treatment (eTNS 120 Hz) or control (eTNS 2 Hz) parameters.
Results:
At entry, subjects were highly drug-resistant, averaging 8.7 seizures per month (treatment group) and 4.8 seizures per month (active controls). On average, subjects failed 3.35 antiepileptic drugs prior to enrollment, with an average duration of epilepsy of 21.5 years (treatment group) and 23.7 years (active control group), respectively. eTNS was well-tolerated. Side effects included anxiety (4%), headache (4%), and skin irritation (14%). The responder rate, defined as >50% reduction in seizure frequency, was 30.2% for the treatment group vs 21.1% for the active control group for the 18-week treatment period (not significant, p = 0.31, generalized estimating equation [GEE] model). The treatment group experienced a significant within-group improvement in responder rate over the 18-week treatment period (from 17.8% at 6 weeks to 40.5% at 18 weeks, p = 0.01, GEE). Subjects in the treatment group were more likely to respond than patients randomized to control (odds ratio 1.73, confidence interval 0.59–0.51). eTNS was associated with reductions in seizure frequency as measured by the response ratio (p = 0.04, analysis of variance [ANOVA]), and improvements in mood on the Beck Depression Inventory (p = 0.02, ANOVA).
Conclusions:
This study provides preliminary evidence that eTNS is safe and may be effective in subjects with DRE. Side effects were primarily limited to anxiety, headache, and skin irritation. These results will serve as a basis to inform and power a larger multicenter phase III clinical trial.
Classification of evidence:
This phase II study provides Class II evidence that trigeminal nerve stimulation may be safe and effective in reducing seizures in people with DRE.
doi:10.1212/WNL.0b013e318285c11a
PMCID: PMC3598453  PMID: 23365066
2.  Docking Phospholipase A2 on Membranes Using Electrostatic Potential–Modulated Spin Relaxation Magnetic Resonance 
Science (New York, N.Y.)  1998;279(5358):1925-1929.
A method involving electron paramagnetic resonance spectroscopy of a site-selectively spin-labeled peripheral membrane protein in the presence and absence of membranes and of a water-soluble spin relaxant (chromium oxalate) has been developed to determine how bee venom phospholipase A2 sits on the membrane. Theory based on the Poisson-Boltzmann equation shows that the rate of spin relaxation of a protein-bound nitroxide by a membrane-impermeant spin relaxant depends on the distance (up to tens of angstroms) from the spin probe to the membrane. The measurements define the interfacial binding surface of this secreted phospholipase A2.
PMCID: PMC3443684  PMID: 9506941
3.  High-throughput computational structure-based characterization of protein families: START domains and implications for structural genomics 
SkyLine, a high-throughput homology modeling pipeline tool, detects and models true sequence homologs to a given protein structure. Structures and models are stored in SkyBase with links to computational function annotation, as calculated by MarkUs. The SkyLine/SkyBase/MarkUs technology represents a novel structure-based approach that is more objective and versatile than other protein classification resources. This structure-centric strategy provides a multidimensional organization and coverage of protein space at the levels of family, function, and genome. The concept of “modelability”, the ability to model sequences on related structures, provides a reliable criterion for membership in a protein family (“leverage”) and underlies the unique success of this approach. The overall procedure is illustrated by its application to START domains, which comprise a Biomedical Theme for the Northeast Structural Genomics Consortium (NESG) as part of the Protein Structure Initiative (PSI). START domains are typically involved in the non-vesicular transport of lipids. While 19 experimentally determined structures are available, the family, whose evolutionary hierarchy is not well determined, is highly sequence diverse, and the ligand-binding potential of many family members is unknown. The SkyLine/SkyBase/MarkUs approach provides significant insights and predicts: 1) many more family members (~4,000) than any other resource; 2) the function for a large number of unannotated proteins; 3) instances of START domains in genomes from which they were thought to be absent; and 4) the existence of two types of novel proteins, those containing dual START domain and those containing N-terminal START domains.
doi:10.1007/s10969-010-9086-7
PMCID: PMC2881152  PMID: 20383749
Homology modeling; Structural genomics; Bioinformatics; Protein function annotation; START domain; Arabidopsis thaliana
4.  Outcome of a Workshop on Applications of Protein Models in Biomedical Research 
Summary
We describe the proceedings and conclusions from a “Workshop on Applications of Protein Models in Biomedical Research” that was held at University of California at San Francisco on 11 and 12 July, 2008. At the workshop, international scientists involved with structure modeling explored (i) how models are currently used in biomedical research, (ii) what the requirements and challenges for different applications are, and (iii) how the interaction between the computational and experimental research communities could be strengthened to advance the field.
doi:10.1016/j.str.2008.12.014
PMCID: PMC2739730  PMID: 19217386
5.  Electrostatic Interactions Drive Membrane Association of the Human Immunodeficiency Virus Type 1 Gag MA Domain▿  
Journal of Virology  2007;81(12):6434-6445.
The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids.
doi:10.1128/JVI.02757-06
PMCID: PMC1900125  PMID: 17392361
6.  Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 DomainD⃞V⃞ 
Molecular Biology of the Cell  2006;17(1):56-66.
The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca2+-binding loops (CBLs) and PIP2 binding site (β-strands 3–4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP2.
doi:10.1091/mbc.E05-06-0499
PMCID: PMC1345646  PMID: 16236797
7.  An Electrostatic Engine Model for Autoinhibition and Activation of the Epidermal Growth Factor Receptor (EGFR/ErbB) Family 
We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca2+] that can activate calmodulin. We postulate the Ca2+/calmodulin complex binds rapidly to residues 645–660 of the juxtamembrane domain, reversing its net charge from +8 to −8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP2), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca2+/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP2; and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.
doi:10.1085/jgp.200509274
PMCID: PMC2266615  PMID: 15955874
8.  Biochemical Characterization of Rous Sarcoma Virus MA Protein Interaction with Membranes 
Journal of Virology  2005;79(10):6227-6238.
The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo.
doi:10.1128/JVI.79.10.6227-6238.2005
PMCID: PMC1091718  PMID: 15858007
9.  The Calcium Binding Loops of the Cytosolic Phospholipase A2 C2 Domain Specify Targeting to Golgi and ER in Live CellsV⃞ 
Molecular Biology of the Cell  2004;15(1):371-383.
Translocation of cytosolic phospholipase A2 (cPLA2) to Golgi and ER in response to intracellular calcium mobilization is regulated by its calcium-dependent lipid-binding, or C2, domain. Although well studied in vitro, the biochemical characteristics of the cPLA2C2 domain offer no predictive value in determining its intracellular targeting. To understand the molecular basis for cPLA2C2 targeting in vivo, the intracellular targets of the synaptotagmin 1 C2A (Syt1C2A) and protein kinase Cα C2 (PKCαC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA2C2 on Syt1C2A and PKCαC2 domain backbones. In response to an intracellular calcium increase, PKCαC2 targeted plasma membrane regions rich in phosphatidylinositol-4,5-bisphosphate, and Syt1C2A displayed a biphasic targeting pattern, first targeting phosphatidylinositol-4,5-bisphosphate-rich regions in the plasma membrane and then the trans-Golgi network. In contrast, the Syt1C2A/cPLA2C2 and PKCαC2/cPLA2C2 hybrids targeted Golgi/ER and colocalized with cPLA2C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA2C2, but not PKCαC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA2C2 and the hybrid domains.
doi:10.1091/mbc.E03-05-0338
PMCID: PMC307554  PMID: 13679516
10.  The Intrinsic Electrostatic Potential and the Intermediate Ring of Charge in the Acetylcholine Receptor Channel 
The Journal of General Physiology  2000;115(2):93-106.
A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction (Imoto et al. 1988). Four of the five subunits in mouse-muscle acetylcholine receptor contribute a glutamate to the ring. These glutamates were mutated to glutamine or lysine, and combinations of mutant and native subunits, yielding net ring charges of −1 to −4, were expressed in Xenopus laevis oocytes. In all complexes, the α subunit contained a Cys substituted for αThr244, three residues away from the ring glutamate αGlu241. The rate constants for the reactions of αThr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2′-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was −230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.
PMCID: PMC2217203  PMID: 10653890
nicotinic; mutagenesis; reaction kinetics; conductance; selectivity

Results 1-10 (10)